Method for cancer cell reprogramming

ABSTRACT

In one embodiment, the invention provides a method of inhibiting cAMP efflux and increasing intracellular cAMP in a subject who suffers from, or who is at risk of developing, a cancer by administering to the subject a therapeutically-effective amount of a cAMP efflux inhibitor. Novel compounds, pharmaceutical compositions, diagnostics and screening methods are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/249,150, filed Apr. 9, 2014, of identical title, now U.S. Pat. No.9,314,460, issued Apr. 19, 2016, which claims priority from U.S.Provisional Patent Application No. 61/810,060, entitled “Method forCancer Cell Reprogramming”, filed Apr. 9, 2013. The complete contents ofthese applications are hereby incorporated by reference in theirentirety herein.

FIELD OF THE INVENTION

In one embodiment, the invention provides a method of inhibiting cAMPefflux and increasing intracellular cAMP in a subject who suffers from,or who is at risk of developing, a cancer by administering to thesubject a therapeutically-effective amount of a cAMP efflux inhibitor.Novel compounds, pharmaceutical compositions, diagnostics and screeningmethods are also provided.

BACKGROUND OF THE INVENTION

One of the breakthroughs in the treatment of leukemia was the discoverythat one of the AML subtypes can be induced to undergo terminaldifferentiation (18). This led to the discovery of all-trans-retinoicacid (ATRA), and the first ATRA-based treatments resulted in completeremission in a large number of cases (26). Consequently, studies aimedat identifying novel approaches that can be employed for differentiationtherapy of AML are considered indispensable (18).

3′-5′-cyclic adenosine monophosphate (cAMP) is implicated in apoptosis,adhesion and differentiation. Multiple hematological malignancies areassociated with a deficiency in apoptosis, and cAMP, the first describedsecond messenger, is implicated in the induction of apoptosis and celldifferentiation(6). A number of researchers have suggested utilizationof the cAMP-related pathways to increase programmed cell death inhematological malignancies(10-12). The pro-apoptotic pathway, promotedby cAMP, depends upon PKA and involves an intrinsicmitochondria-dependent mechanism. Multiple (Bcl)-related family proteinsthat include pro-apoptotic Bax, Bak, Bad, Bim and anti-apoptotic (suchas Bcl-2) proteins are implicated in the release of cytochrome c andSmac (second mitochondria-derived activator of caspases/DIABLO (directIAP-binding protein with low pI). An increased expression of the Bimprotein in response to cAMP/PKA-induced apoptosis was required forinduction of G1 phase cell cycle arrest and apoptosis(27). However, thesignaling pathway leading from the elevation of cAMP to apoptotic celldeath is still under active investigation, and the exact therapeutictargets can be cell-type specific and may vary from patient to patient.

The loss of integrin-dependent adhesion is one of the early hallmarks inthe development of precancerous lesions. In solid tumors, such as breastor ovarian cancer, integrins provide a signal that maintains cellattachment to the basal membrane, and supports cell polarization. Lossof beta 1 integrin-dependent adhesion results in a perturbation ofnormal cell morphology, and can play a role in tumor development.Furthermore, mobilization of hematopoietic progenitors/blasts into theperipheral blood, which is dependent on the alpha 4 beta 1 integrin(VLA-4)(9), can correlate with more aggressive disease in certainleukemias(2). Our recent discovery that cyclic nucleotides (cAMP(4), and3′-5′-cyclic guanosine monophosphate (cGMP)(3)) can actively downregulate VLA-4 integrin ligand-binding affinity, rapidly down-modulatecell adhesion, and possibly, induce mobilization of hematopoieticprogenitors suggested a specific role of these cyclic nucleotides inhematological malignancies.

Furthermore, cAMP is shown to play an important role in leukemic celldifferentiation. The cell-permeable analog of cAMP inducesdifferentiation in the human promonocytic U937 cell line(23; 24). Thecytoplasmic level of cAMP in this cell line was modulated through the H2histamine receptor, which at the same time can induce VLA-4 integrinde-activation and cell de-adhesion(4). Thus, it is plausible thatmodulation of the cAMP level, rather than targeting individual proteinsdownstream of the cAMP signal, can be utilized as a tool for celldifferentiation therapy.

cAMP is synthesized by the family of enzymes termed adenylate cyclases(adenylyl cyclases, ACs). Enzyme activity is controlled by two classesof GPCRs: GalphaS-coupled stimulate cyclase activity, andGalphaI-coupled inhibit the enzyme. Next, cAMP can be hydrolyzed by thesuperfamily of enzymes called 3′,5′-cyclic nucleotide phosphodiesterases(PDEs), which can have different specificities (cAMP vs. cGMP),localization, and regulation. PDEs are accepted targets for treatment ofhematological malignancies, and a number of PDE inhibitors are beingtested in different model systems (10).

Another underappreciated mechanism is the removal of the cyclicnucleotides from the cytoplasm by the ATP-binding cassette transporter(ABC-transporter) family of proteins. According to the UCSF-FDATransPortal database only two transporters, MRP4 and MRP5 are implicatedin the active removal of cAMP. Moreover, MRP4 is expressed on the plasmamembrane of CD34+ cells, exhibits higher binding affinity for cAMP (vs.cGMP), and the expression of MRP4 (but not MRP5) significantly decreasesduring leukocyte differentiation (14). This suggests thatun-differentiated cell phenotypes can have an increased ability toremove cAMP from the cytoplasm. We recognize that other transporters mayparticipate in this process.

Several clinical studies point toward a specific role of cyclicnucleotides in patients with leukemia. In the urine of patients withfour types of leukemia (AML, CML, ALL, and CLL) the concentration andurinary excretion of cyclic nucleotides was higher than in healthyvolunteers, with the largest difference between acute leukemia patientsand control groups (22). In addition, the plasma level of cyclicnucleotides correlated with the stage of the disease, and it wasdifferent in patients who attained remission vs. relapsed individuals(17). At the same time, in WBCs, the cAMP concentration in leukemiccells was lower than in normal cells (16; 22). Based on these and otherdata we proposed that certain leukemic cells have developed a mechanismthat actively removes the pro-apoptotic second messenger (cAMP) fromcells into the blood, thus protecting the cell from apoptosis. SincecAMP is excreted through the kidneys, this mechanism explains the lowerintracellular cAMP content in leukemic cells (cAMP is continuouslyremoved), the higher plasma and urine concentrations, and thecorrelation between cAMP concentration and disease progression.

Several recent reviews discuss cyclic nucleotide modulation as apossible option for cancer therapy. Because overexpression of PDEisoforms has been described in several cancers, PDE inhibitors areenvisioned as a viable option to restore normal nucleotide metabolism(10; 21). Downstream effectors of the cAMP/PKA-induced apoptotic pathway(such as the Bim protein) are also under investigation for targeting incancer (1). The interest in cAMP and cGMP efflux in the cancer field wasstimulated mainly by the fact that cyclic nucleotides represent naturalsubstrates for multidrug resistance proteins (MRPs/ABCCs), implicated inthe efflux of anti-cancer drugs, and not usually envisioned as amechanism for modulating the signaling for cell reprogramming.

The idea that cell “maturation”, resistant to ATRA-induceddifferentiation, can be promoted by cAMP-elevating agents, or by usingcAMP analogs, is nearly twenty years old (20). An increase in cellularcAMP reduces the effective concentration of ATRA required to achievematuration(19). The role of the cAMP pathway in t(15; 17) APL has beenstudied for many years. They uncovered cross-talk between arsenictrioxide and cAMP signaling (29), described the rapid increase in cAMPand PKA expression after ATRA treatment (28), and showed the benefits ofthe ATRA/arsenic trioxide combination for therapy of APL (25). Severalrecent reports highlight the role of cAMP/PKA-signaling for celldifferentiation. The cAMP analog/ATRA combination is shown to improvethe differentiation of t(11; 17)(q23;q21) APL cells, the subset carryingPLZF/RARa fusion that poorly responds to ATRA (7). The activation of thetwo PKA isozymes is required for ATRA-induced maturation of APL cells(13). Thus, our attention to the mechanisms, modulating cAMP levels iswell justified. A seemingly surprising result that the treatment ofblasts with cAMP-elevating agents protects cell from cytotoxic drugs(5), can be also interpreted according to our hypothesis: the same classof proteins, which is implicated in the cAMP regulation, can alsomediate drug resistance.

SUMMARY OF THE INVENTION

The present invention is directed to the unexpected discovery thatcancer cells can be reprogrammed by initiating apoptotic escape byblocking the removal of the second messenger in a cancer cell. To thatend, a number of compounds have been identified which show activity asinhibitors of cAMP efflux. These compounds show activity as inhibitorsof cAMP efflux and consequently, are identified as inhibitors of cancercell apoptotic escape, one of the principal mechanisms which enabletumors to grow and elaborate. Further, the compounds are effectiveanticancer agents which find use as inhibitors of the removal of thesecond messenger in cancer cells, resulting in restoration of theapoptosis of cancer cells as well as treatment and prevention of cancer,especially including metastasis of cancer, through a novel mechanism.The compounds may be used alone or in combination with other anticanceragents to treat and/or reduce the likelihood of cancer, includingespecially metastatic cancer.

Compounds according to the present invention may also be used as ligandsin assays which are used to identify compounds which exhibit activity asinhibitors of cAMP efflux and as agents to restore apoptosis in cancercells.

In one aspect of our invention, we have discovered novelsmall-molecule-based methods of reprogramming a population of cancercells to reestablish an apoptotic escape. In one aspect of ourinvention, compounds which exhibit activity as inhibitors of cAMP effluxreprogram cancer cells, thereby restoring an anti-apoptotic mechanismand inhibiting and treating cancer, including metastatic cancer.

Exemplary compounds which find use in the present invention include thefollowing compounds (most of which are also set forth in FIG. 1,attached hereto):

harmalol, artenisinin and artemether or a pharmaceutically acceptablesalt, stereoisomer (including diastereomers and/or enantiomers), solvateor polymorph thereof.

In further embodiments, the present invention also relates to methodsfor treating or reducing the likelihood of cancer or reducing thelikelihood of metastasis in a patient in need, comprising administeringa therapeutically-effective amount of one or more inhibitors of cAMPefflux, e.g. at least one compound selected from the group consisting of

harmalol, artenisinin and artemether or a pharmaceutically acceptablesalt, stereoisomer (which term includes diastereomers and enantiomers),solvate or polymorph thereof, in combination with a pharmaceuticallyacceptable carrier, additive and/or excipient and optionally, at leastone additional anticancer agent.

In still additional embodiments, the present invention relates tomethods of reprogramming cancer cells to reestablish an apoptoticmechanism comprising exposing said cancer cells to atherapeutically-effective amount of one or more inhibitors of cAMPefflux, e.g. at least one compound selected from the group consisting of

harmalol, artenisinin and artemether or a pharmaceutically acceptablesalt, stereoisomer, solvate or polymorph thereof, in combination with apharmaceutically acceptable carrier, additive and/or excipient andoptionally, at least one additional anticancer agent.

One embodiment of our invention provides a method of inhibiting cAMPefflux and increasing intracellular cAMP in a subject who suffers from,or who is at risk of developing, a cancer, the method comprisingadministering to the subject a therapeutically-effective amount of acAMP efflux inhibitor, preferably a compound selected from the groupconsisting of artemisinin, artemether, artesunate, dihydroartemisinin,patulin, pyrithione zinc, parthenolide, quinalizarin, clioquinol,cryptotanshinone and harmalol, and the pharmaceutically-acceptablesalts, stereoisomers, solvates and polymorphs thereof.

In a preferred embodiment, the subject suffers from one or more cancersselected from the group consisting of kidney cancer, oral squamous cellcarcinoma, glioblastoma, colon cancer, colorectal cancer andhematological cancer and is administered a therapeutically-effectiveamount of patulin or a pharmaceutically-acceptable salt, stereoisomer,solvate or polymorph thereof.

In another preferred embodiment, the subject suffers from one or morecancers selected from the group consisting of melanoma, cervical cancer,hematological cancer, breast cancer and cystic fibrosis and isadministered a therapeutically-effective amount of parthenolide or apharmaceutically-acceptable salt, stereoisomer, solvate or polymorphthereof.

In another preferred embodiment, the subject suffers from one or morecancers selected from the group consisting of ovarian cancer, cervicalcancer, breast cancer, liver cancer, melanoma, pancreatic cancer, lungcancer, hematological cancer, prostate cancer, gioma and osteosacoma andis administered a therapeutically-effective amount of artesunate and/ordihydroartemisinin, or a pharmaceutically-acceptable salt, stereoisomer,solvate or polymorph thereof.

In another preferred embodiment, the subject suffers from one or morecancers selected from the group consisting of hematological cancer,breast cancer HeLa, prostate cancer, hematological cancer, melanoma,lung cancer and rhabodomyosarcoma and is administered atherapeutically-effective amount of clioquinol and/or cryptotanshinoneor a pharmaceutically-acceptable salt, stereoisomer, solvate orpolymorph thereof.

In another embodiment, the invention provides a pharmaceutical compoundthat inhibits cAMP efflux and increases intracellular cAMP, thecomposition comprising:

(a) a therapeutically-effective amount of a cAMP efflux inhibitorselected from the group consisting of artemisinin, artemether,artesunate, dihydroartemisinin, patulin, pyrithione zinc, parthenolide,quinalizarin, clioquinol, cryptotanshinone and harmalol, and thepharmaceutically-acceptable salts, stereoisomers, solvates andpolymorphs thereof; and, optionally(b) one or more additional anti-cancer agents and/or apharmaceutically-acceptable excipient.

In other embodiments, the subject who suffers from one or more of theaforementioned cancers is treated concomitantly by a cAMP effluxinhibitor, one or more additional anti-cancer agents as described hereinand, optionally, radiotherapy.

In another embodiment, the invention provides a diagnostic method fordetermining whether a subject suffers from one or more cancers, themethod comprising:

(a) assaying a sample obtained from the subject for levels of cAMPefflux and intracellular cAMP; and

(b) predicting an increased likelihood that the subject suffers from oneor more cancers upon detection of elevated levels of cAMP efflux andintracellular cAMP.

In another embodiment, the invention provides a method of determiningwhether a composition is effective in the treatment of one or morecancers, the method comprising contacting a eukaryotic cell sample withthe composition, measuring cellular cAMP efflux, and comparing measuredcellular cAMP efflux with levels of cAMP efflux in a control eukaryoticcell sample, wherein reduced expression levels of cAMP efflux whencompared to control expression levels indicates that the composition iseffective in the treatment of one or more cancers.

In a preferred embodiment, the invention provides a diagnostic methodfor determining whether a subject suffers from one or more cancers, themethod comprising:

(a) assaying a sample obtained from the subject for levels of cAMPefflux and intracellular cAMP by contacting the sample with afluorescent cAMP analog (F-cAMP) in a flow cytometric assay to monitorcAMP efflux; and

(b) predicting an increased likelihood that the subject suffers from oneor more cancers upon detection of elevated levels of cAMP efflux andintracellular cAMP.

The methods and formulations described herein prove particularlyeffective in treating a wide variety of cancers that have beenpreviously been associated with high rates of remission and poorlong-term survival.

These and other aspects of the invention are described further in theDetailed Description of the Invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows compounds which exhibit activity as inhibitors of cAMPefflux and are useful in the present invention for reprogramming cancercells, restoring an anti-apoptotic mechanism and in inhibiting andtreating cancer, including metastatic cancer.

FIG. 2 shows the results of an assay for detecting cyclic AMP efflux.Cells were loaded with the Alexa488-cAMP analog (top). After incubation(16 hours) green fluorescence was measured using a flow cytometer.MK-571 was previously shown to block cAMP efflux, and it has been usedas control (bottom).

FIG. 2A shows a proposed model for the experiments described in theExamples herein. Somatic cells experience genetic instability, whichgenerates mutations, thus increasing intracellular cAMP. To evade theintrinsic apoptosis induced by elevated intracellular cAMP, cancer cellsefflux cAMP, releasing the second messenger into the blood and urine.

FIG. 3 shows the cell viability as determined using CellTiter-Glo®assay. Cells were incubated for 24 hours with increasing concentrationsof compounds identified in the cAMP efflux assay (FIG. 1).

FIG. 4 shows the percent of Annexin V and 7-AAD double positive eventsplotted vs. the concentration of cAMP efflux blockers. Cells wereincubated with increasing concentrations of compounds identified in cAMPefflux assay, and assayed for binding of Annexin V and 7-AAD.

FIG. 4A shows the effects of identified cAMP efflux inhibitors on U937cell cycle after overnight incubation. B) Bar graph indicatingpercentages of cells gated in each phase of the cell cycle afterdose-dependent treatment with cAMP efflux inhibitors. Gating was done asfollows: G1/G0: DNA=2n, S: 2n<DNA<4n, G2/M: DNA=4n, Apoptosis (A):DNA<2n. C) Calculated EC50 concentrations of hit compounds for apoptoticevents shown in (B). EC50 values were determined by variable slope log(agonist) vs response fits with the following constraints: top=100,bottom=0.

FIG. 5 shows the viability of human PBMCs (top) and U937 cells (bottom)treated side by side with different concentrations of the cAMP effluxblocking compounds determined using the CellTiter-Glo® assay.

FIG. 5A. Effects of identified cAMP efflux inhibitors on U937proliferation. Cells were stained with CFSE in bulk, and then separatedinto cultures containing either solvent, 3 μM, or 10 μM compounds.Samples were taken from cultures after 48, 72, and 96 hours afterinitial culture time. A and B) Raw data of CFSE MFI in cells remainingafter treatment with hit compounds, as measured by flow cytometry.Decreases in CFSE MFI over time indicate cell proliferation. Data in (B)were analyzed by one-way ANOVA with repeated measures with a Dunnettpost test to compare treated samples to DMSO control values (n=3,*p<0.05, **p<0.01, ***p<0.001). Data shown are the result of 3independent experiments.

FIG. 6. Removal of cAMP-Alexa 488 probe from different hematopoieticcell lines. Cell autofluorescence was subtracted. The data werenormalized to the initial cAMP-fluorescence value. The graph showsremaining fluorescent probe. Therefore, the lowest signal corresponds tothe higher probe removal activity.

FIG. 6A. Effects of identified cAMP efflux inhibitors on cell viabilityof U937, normal human PBMCs, and the B-lineage ALL cell lines 697, Reh,MHH Call 3, RS4; 11, Sup B15, and Nalm 6. Data were normalized such that1% DMSO negative control was equal to 100% viability. Data for U937 andPBMCs were fit using variable slope log (agonist) vs response nonlinearregressions with the following constraints: top=100, bottom=0. Data forB-ALL cell lines were fit using variable slope sigmoidal dose responsefits with the following constraints: top=100, bottom=0, hill slope=1. B)EC50 values determined from fits described in (A) for tested compoundswith each cell type.

FIG. 7. cAMP accumulation (A), cell viability (B), and a crosscorrelation between EC50s for cAMP accumulation and cell viability forREH cells. Cells were treated with different concentrations of compoundhits. Dose response curves were generated using Graphpad Prism software.

FIG. 7A. Retention of a fluorescent cAMP analog (F-cAMP) inhematopoietic cell lines after overnight incubation. A) F-cAMP leakagefrom B-lineage ALL cell lines after overnight incubation at 4° C., or37° C., and in the presence of the HTS positive control/known cAMPefflux inhibitor, MK-571. Cell autofluorescence was subtracted, and thedata were normalized to the cAMP-fluorescence value after initialstaining. The graph depicts F-cAMP remaining within cells, andtherefore, lower values correspond to higher probe removal activity. B)A negative correlation occurred between cAMP-Alexa 488 fluorescencewhich remained in cells incubated at 37° C. alone and in the presence ofcAMP efflux inhibitor MK-571. Simply, cell lines which poorly removedcAMP were best inhibited by MK-571, and those cell lines which activelyremoved most of the incorporated cAMP were less inhibited by MK-571.Each cell line is represented by three independently processed samplesfor each treatment variant. C) F-cAMP retention after overnightincubation with HTS-identified compounds in dose response, as measuredby flow cytometry for mean channel intensity (MCI). Percentage valuesare relative to fluorescence values of negative control cells incubatedat 4° C. Note that there are different scales on the y-axes of thegraphs. Lines indicate variable slope sigmoidal dose response fits withthe following constraints: top=100, bottom=0, hill slope=1. D)Calculated EC50 concentrations of tested compounds with each cell linebased on fits from (B). Data for the U937 cell line were collected atthe time of HTS hit compound validation, and analyzed in the samemanner.

FIG. 8. Determination of ATP-binding cassette transporter ABCC4(ABCC4)-specific binding sites on B-lineage ALL cell lines. ABCC4 is amembrane transporter implicated in cAMP efflux. Specific binding siteswere deduced by binding of primary ABCC4 antibody and fluorescentsecondary antibody, and analyzed by flow cytometry. Calibration beadsallowed mean channel intensity (MCI) values to be converted intonon-specific binding sites. Binding site values from IgG primaryantibody-bound isotype control cells were subtracted from the ABCC4non-specific binding site data to calculate the number of ABCC4-specificbinding sites per cell.

FIG. 9. Accumulation of intracellular cAMP after overnight treatmentwith identified cAMP efflux inhibiting compounds. A) Dose-dependenteffects of hit compounds on intracellular cAMP concentrations inB-lineage ALL cell lines, as measured by the cAMP-Glo luminescenceassay. Relative luminescence values (RLU) were normalized based on 1%DMSO control and 1.53 nM concentration wells for each cell line andtreatment. Lines indicate variable slope sigmoidal dose response fitswith constraint of hill slope=1. B The relationship between increasedintracellular cAMP (i) and decreased cell viability (ii), as evidencedwith the Reh cell line after overnight treatment with HTS-identifiedcAMP efflux inihibitors. iii) Comparison of compound EC50 values fromboth the viability and cAMP accumulation assays in Reh cells; r²=0.9776.

DETAILED DESCRIPTION OF THE INVENTION

The following terms are used throughout the specification to describethe present invention. Where a term is not given a specific definitionherein, that term is to be given the same meaning as understood by thoseof ordinary skill in the art. The definitions given to the diseasestates or conditions which may be treated using one or more of thecompounds according to the present invention are those which aregenerally known in the art.

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” include plural referents unlessexpressly and unequivocally limited to one referent. Thus, for example,reference to “a compound” includes two or more different compounds. Asused herein, the term “include” and its grammatical variants areintended to be non-limiting, such that recitation of items in a list isnot to the exclusion of other like items that can be substituted orother items that can be added to the listed items.

The term “patient” or “subject” is used throughout the specification todescribe an animal, preferably a human, to whom treatment, includingprophylactic treatment, with the compositions according to the presentinvention is provided (a patient or subject in need). For treatment ofthose infections, conditions or disease states which are specific for aspecific animal such as a human patient, the term patient refers to thatspecific animal. In many instances, methods are applied to patients orsubjects who are suspected of having cancer, or who have cancer and aresuspected of having the cancer metastasize.

The term “compound” is used herein to refer to any specific chemicalcompound disclosed herein. Within its use in context, the term generallyrefers to a single small molecule as disclosed herein, but in certaininstances may also refer to other forms of the compound, includingpharmaceutically acceptable salts, stereoisomers, includingdiastereomers and enantiomers, solvates and polymorphs. The termcompound includes active metabolites of compounds and/orpharmaceutically acceptable salts thereof.

The term “effective amount” is used throughout the specification todescribe concentrations or amounts of formulations or other componentswhich are used in amounts, within the context of their use, to producean intended effect according to the present invention, in this case toreprogram cancer cells by inhibiting efflux of cAMP or to otherwiseincrease cAMP in cancer cells and restore an apoptotic mechanism to thecell. The formulations or component may be used to produce a favorablechange in a disease or condition treated, whether that change is aremission of effects of a disease state or condition, a favorablephysiological result, a reversal or attenuation of a disease state orcondition treated, the prevention or the reduction in the likelihood ofa condition or disease-state occurring, depending upon the disease orcondition treated, especially cancer and metastatic cancer. Whereformulations are used in combination, each of the formulations is usedin an effective amount, wherein an effective amount may include asynergistic amount. The amount of formulation used in the presentinvention may vary according to the nature of the formulation, the ageand weight of the patient and numerous other factors which may influencethe bioavailability and pharmacokinetics of the formulation, the amountof formulation which is administered to a patient generally ranges fromabout 0.001 mg/kg to about 50 mg/kg or more, about 0.5 mg/kg to about 25mg/kg, about 0.1 to about 15 mg/kg, about 1 mg to about 10 mg/kg per dayand otherwise described herein. The person of ordinary skill may easilyrecognize variations in dosage schedules or amounts to be made duringthe course of therapy.

The term “prophylactic” is used to describe the use of a formulationdescribed herein which reduces the likelihood of an occurrence of acondition or disease state in a patient or subject. The term “reducingthe likelihood” refers to the fact that in a given population ofpatients, the present invention may be used to reduce the likelihood ofan occurrence, recurrence or metastasis of disease in one or morepatients within that population of all patients, rather than prevent, inall patients, the occurrence, recurrence or metastasis of a diseasestate, in this case cancer. In preferred aspects of the invention,compounds according to the present invention may be used to reduce thelikelihood of cancer, including metastasis of cancer.

The term “pharmaceutically acceptable” refers to a salt form or otherderivative (such as an active metabolite or prodrug form) of the presentcompounds or a carrier, additive or excipient which is not unacceptablytoxic to the subject to which it is administered.

“Treat”, “treating”, and “treatment”, etc., as used herein, refer to anyaction providing a benefit to a patient at risk for or afflicted with adisease, including improvement in the condition through lessening orsuppression of at least one symptom, delay in progression of thedisease, prevention or delay in the onset of the disease, etc.Treatment, as used herein, encompasses both prophylactic and therapeutictreatment.

The term “neoplasia” refers to the uncontrolled and progressivemultiplication of tumor cells, under conditions that would not elicit,or would cause cessation of, multiplication of normal cells. Neoplasiaresults in a “neoplasm”, which is defined herein to mean any new andabnormal growth, particularly a new growth of tissue, in which thegrowth of cells is uncontrolled and progressive. Thus, neoplasiaincludes “cancer”, which herein refers to a proliferation of tumor cellshaving the unique trait of loss of normal controls, resulting inunregulated growth, lack of differentiation, local tissue invasion,and/or metastasis.

As used herein, neoplasms include, without limitation, morphologicalirregularities in cells in tissue of a subject or host, as well aspathologic proliferation of cells in tissue of a subject, as comparedwith normal proliferation in the same type of tissue. Additionally,neoplasms include benign tumors and malignant tumors (e.g., colontumors) that are either invasive or noninvasive. Malignant neoplasms aredistinguished from benign neoplasms in that the former show a greaterdegree of anaplasia, or loss of differentiation and orientation ofcells, and have the properties of invasion and metastasis. Examples ofneoplasms or neoplasias from which the target cell of the presentinvention may be derived include, without limitation, carcinomas (e.g.,squamous-cell carcinomas, adenocarcinomas, hepatocellular carcinomas,and renal cell carcinomas), particularly those of the bladder, bowel,breast, cervix, colon, esophagus, head, kidney, liver, lung, neck,ovary, pancreas, prostate, and stomach; leukemias; benign and malignantlymphomas, particularly Burkitt's lymphoma and Non-Hodgkin's lymphoma;benign and malignant melanomas; myeloproliferative diseases; sarcomas,particularly Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma,liposarcoma, myosarcomas, peripheral neuroepithelioma, and synovialsarcoma; tumors of the central nervous system (e.g., gliomas,astrocytomas, oligodendrogliomas, ependymomas, gliobastomas,neuroblastomas, ganglioneuromas, gangliogliomas, medulloblastomas,pineal cell tumors, meningiomas, meningeal sarcomas, neurofibromas, andSchwannomas); germ-line tumors (e.g., bowel cancer, breast cancer,prostate cancer, cervical cancer, uterine cancer, lung cancer, ovariancancer, testicular cancer, thyroid cancer, astrocytoma, esophagealcancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer,and melanoma); mixed types of neoplasias, particularly carcinosarcomaand Hodgkin's disease; and tumors of mixed origin, such as Wilms' tumorand teratocarcinomas (Beers and Berkow (eds.), The Merck Manual ofDiagnosis and Therapy, 17.sup.th ed. (Whitehouse Station, N.J.: MerckResearch Laboratories, 1999) 973-74, 976, 986, 988, 991.

The term “additional anticancer agent” shall mean chemotherapeuticagents such as an agent selected from the group consisting ofmicrotubule-stabilizing agents, microtubule-disruptor agents, alkylatingagents, antimetabolites, epidophyllotoxins, antineoplastic enzymes,topoisomerase inhibitors, inhibitors of cell cycle progression, andplatinum coordination complexes. These may be selected from the groupconsisting of everolimus, trabectedin, abraxane, TLK 286, AV-299,DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244(ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin,vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, aFLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurorakinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDACinhbitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFRTK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinaseinhibitors, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mek)inhibitor, a VEGF trap antibody, pemetrexed, erlotinib, dasatanib,nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu,nolatrexed, azd2171, batabulin, ofatumumab, zanolimumab, edotecarin,tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab,ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490,cilengitide, gimatecan, IL13-PE38QQR, INO 1001, IPdR₁ KRX-0402,lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102,talampanel, atrasentan, Xr 311, romidepsin, ADS-100380, sunitinib,5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin,liposomal doxorubicin, 5′-deoxy-5-fluorouridine, vincristine,temozolomide, ZK-304709, seliciclib; PD0325901, AZD-6244, capecitabine,L-Glutamic acid,N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-,disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan,tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole,DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen,bevacizumab, IMC-1C11, CHIR-258,);3-[5-(methylsulfonylpiperadinemethyl)-indolylj-quinolone, vatalanib,AG-013736, AVE-0005, the acetate salt of [D-Ser(But)6, Azgly10](pyro-Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH₂ acetate[C₅₉H₈₄N₁₈Oi₄-(C₂H₄O₂)_(X) where x=1 to 2.4], goserelin acetate,leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate,hydroxyprogesterone caproate, megestrol acetate, raloxifene,bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714;TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody,erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib, BMS-214662,tipifarnib; amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid,valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951,aminoglutethimide, amsacrine, anagrelide, L-asparaginase, BacillusCalmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan,carboplatin, carmustine, chlorambucil, cisplatin, cladribine,clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, diethylstilbestrol, epirubicin, fludarabine,fludrocortisone, fluoxymesterone, flutamide, gemcitabine, hydroxyurea,idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine,mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate,mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin,pamidronate, pentostatin, plicamycin, porfimer, procarbazine,raltitrexed, rituximab, streptozocin, teniposide, testosterone,thalidomide, thioguanine, thiotepa, tretinoin, vindesine,13-cis-retinoic acid, phenylalanine mustard, uracil mustard,estramustine, altretamine, floxuridine, 5-deooxyuridine, cytosinearabinoside, 6-mecaptopurine, deoxycoformycin, calcitriol, valrubicin,mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat,COL-3, neovastat, BMS-275291, squalamine, endostatin, SU5416, SU6668,EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene,idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab,denileukin diftitox, gefitinib, bortezimib, paclitaxel, cremophor-freepaclitaxel, docetaxel, epithilone B, BMS-247550, BMS-310705,droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene,fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339,ZK186619, topotecan, PTK787/ZK 222584, VX-745, PD 184352, rapamycin,40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001,ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646,wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin,erythropoietin, granulocyte colony-stimulating factor, zolendronate,prednisone, cetuximab, granulocyte macrophage colony-stimulating factor,histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylatedinterferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase,lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane,alemtuzumab, all-transretinoic acid, ketoconazole, interleukin-2,megestrol, immune globulin, nitrogen mustard, methylprednisolone,ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine,bexarotene, tositumomab, arsenic trioxide, cortisone, editronate,mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase,strontium 89, casopitant, netupitant, an NK-1 receptor antagonists,palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide,lorazepam, alprazolam, haloperidol, droperidol, dronabinol,dexamethasone, methylprednisolone, prochlorperazine, granisetron,ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin,epoetin alfa and darbepoetin alfa, among others.

A “pharmaceutically acceptable salt” of the present compound generallyrefers to pharmaceutically acceptable salts form of a compound which canform a salt, because of the existence of for example, amine groups,carboxylic acid groups or other groups which can be ionized in a sampleacid-base reaction. A pharmaceutically acceptable salt of an aminecompound, such as those contemplated in the current invention, include,for example, ammonium salts having as counterion an inorganic anion suchas chloride, bromide, iodide, sulfate, sulfite, nitrate, nitrite,phosphate, and the like, or an organic anion such as acetate, malonate,pyruvate, propionate, fumarate, cinnamate, tosylate, and the like.Certain compounds according to the present invention which havecarboxylic acid groups or other acidic groups which may formpharmaceutically acceptable salts, for example, as carboxylate salts(sodium, potassium, magnesium, zinc, etc.), are also contemplated by thepresent invention.

Formulations of the invention may include a pharmaceutically acceptablediluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.Acceptable formulation materials preferably are nontoxic to recipientsat the dosages and concentrations employed. The pharmaceuticalformulations may contain materials for modifying, maintaining orpreserving, for example, the pH, osmolarity, viscosity, clarity, color,isotonicity, odor, sterility, stability, rate of dissolution or release,adsorption or penetration of the composition. Suitable formulationmaterials include, but are not limited to, amino acids (such as glycine,glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants(such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite);buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates orother organic acids); bulking agents (such as mannitol or glycine);chelating agents (such as ethylenediamine tetraacetic acid (EDTA));complexing agents (such as caffeine, polyvinylpyrrolidone,beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers;monosaccharides, disaccharides, and other carbohydrates (such asglucose, mannose or dextrins); proteins (such as serum albumin, gelatinor immunoglobulins); coloring, flavoring and diluting agents;emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone);low molecular weight polypeptides; salt-forming counterions (such assodium); preservatives (such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide);solvents (such as glycerin, propylene glycol or polyethylene glycol);sugar alcohols (such as mannitol or sorbitol); suspending agents;surfactants or wetting agents (such as pluronics, polyethylene glycol(PEG), sorbitan esters, polysorbates such as polysorbate 20 andpolysorbate 80, Triton, trimethamine, lecithin, cholesterol, ortyloxapal); stability enhancing agents (such as sucrose or sorbitol);tonicity enhancing agents (such as alkali metal halides, preferablysodium or potassium chloride, mannitol, or sorbitol); delivery vehicles;diluents; excipients and/or pharmaceutical adjuvants. See, for example,REMINGTON'S PHARMACEUTICAL SCIENCES, 18.sup.th Edition, (A. R. Gennaro,ed.), 1990, Mack Publishing Company.

Optimal pharmaceutical formulations can be determined by one skilled inthe art depending upon, for example, the intended route ofadministration, delivery format and desired dosage. See, for example,REMINGTON'S PHARMACEUTICAL SCIENCES, Id. Such formulations may influencethe physical state, stability, rate of in vivo release and rate of invivo clearance of the antibodies of the invention.

Primary vehicles or carriers in a pharmaceutical formulation caninclude, but are not limited to, water for injection, physiologicalsaline solution or artificial cerebrospinal fluid, possibly supplementedwith other materials common in compositions for parenteraladministration. Neutral buffered saline or saline mixed with serumalbumin are further exemplary vehicles. Pharmaceutical formulations cancomprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH4.0-5.5, which may further include sorbitol or a suitable substitute.Pharmaceutical formulations of the invention may be prepared for storageby mixing the selected composition having the desired degree of puritywith optional formulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES,Id.) in the form of a lyophilized cake or an aqueous solution. Further,the formulations may be formulated as a lyophilizate using appropriateexcipients such as sucrose.

Formulation components are present in concentrations that are acceptableto the site of administration. Buffers are advantageously used tomaintain the composition at physiological pH or at a slightly lower pH,typically within a pH range of from about 5 to about 8.

The pharmaceutical formulations of the invention can be deliveredparenterally. When parenteral administration is contemplated, thetherapeutic formulations for use in this invention may be in the form ofa pyrogen-free, parenterally acceptable aqueous solution. Preparationinvolves the formulation of the desired immunomicelle, which may providecontrolled or sustained release of the product which may then bedelivered via a depot injection. Formulation with hyaluronic acid hasthe effect of promoting sustained duration in the circulation.

Formulations may be formulated for inhalation. In these embodiments, astealth immunomicelle formulation is formulated as a dry powder forinhalation, or inhalation solutions may also be formulated with apropellant for aerosol delivery, such as by nebulization. Pulmonaryadministration is further described in PCT Application No.PCT/US94/001875, which describes pulmonary delivery of chemicallymodified proteins and is incorporated by reference.

Formulations of the invention can be delivered through the digestivetract, such as orally. The preparation of such pharmaceuticallyacceptable compositions is within the skill of the art. Formulationsdisclosed herein that are administered in this fashion may be formulatedwith or without those carriers customarily used in the compounding ofsolid dosage forms such as tablets and capsules. A capsule may bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized Additional agents can be includedto facilitate absorption. Diluents, flavorings, low melting point waxes,vegetable oils, lubricants, suspending agents, tablet disintegratingagents, and binders may also be employed.

A formulation may involve an effective quantity of a microparticlecontaining formulation as disclosed herein in a mixture with non-toxicexcipients that are suitable for the manufacture of tablets. Bydissolving the tablets in sterile water, or another appropriate vehicle,solutions may be prepared in unit-dose form. Suitable excipientsinclude, but are not limited to, inert diluents, such as calciumcarbonate, sodium carbonate or bicarbonate, lactose, or calciumphosphate; or binding agents, such as starch, gelatin, or acacia; orlubricating agents such as magnesium stearate, stearic acid, or talc.

The pharmaceutical composition to be used for in vivo administrationtypically is sterile. In certain embodiments, this may be accomplishedby filtration through sterile filtration membranes. In certainembodiments, where the composition is lyophilized, sterilization usingthis method may be conducted either prior to or following lyophilizationand reconstitution. In certain embodiments, the composition forparenteral administration may be stored in lyophilized form or in asolution. In certain embodiments, parenteral compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Once the formulation of the invention has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or as a dehydrated or lyophilized powder. Such formulations may bestored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration.

Administration routes for formulations of the invention include orally,through injection by intravenous, intraperitoneal, intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,intra-ocular, intraarterial, intraportal, or intralesional routes; bysustained release systems or by implantation devices. The pharmaceuticalformulations may be administered by bolus injection or continuously byinfusion, or by implantation device. The pharmaceutical formulationsalso can be administered locally via implantation of a membrane, spongeor another appropriate material onto which the desired molecule has beenabsorbed or encapsulated. Where an implantation device is used, thedevice may be implanted into any suitable tissue or organ, and deliveryof the desired molecule may be via diffusion, timed-release bolus, orcontinuous administration.

A “control” as used herein may be a positive or negative control asknown in the art and can refer to a HLTF agonist or antagonistcomposition (e.g. small molecule), or a control cell, tissue, sample, orsubject. The control may, for example, be examined at precisely ornearly the same time the test cell, tissue, sample, or subject isexamined. The control may also, for example, be examined at a timedistant from the time at which the test cell, tissue, sample, or subjectis examined, and the results of the examination of the control may berecorded so that the recorded results may be compared with resultsobtained by examination of a test cell, tissue, sample, or subject. Forinstance, as can be appreciated by a skilled artisan, a control maycomprise data from one or more control subjects that is stored in areference database. The control may be a subject who is similar to thetest subject (for instance, may be of the same gender, same race, samegeneral age and/or same general health) but who is known to not have afibrotic disease. As can be appreciated by a skilled artisan, themethods of the invention can also be modified to compare a test subjectto a control subject who is similar to the test subject (for instance,may be of the same gender, same race, same general age and/or samegeneral health) but who is known to express symptoms of a disease. Inthis embodiment, a diagnosis of a disease or staging of a disease can bemade by determining whether protein or gene expression levels asdescribed herein are statistically similar between the test and controlsubjects.

As described in U.S. Pat. No. 7,908,091, “the term “profile” includesany set of data that represents the distinctive features orcharacteristics associated with a tumor, tumor cell, and/or cancer. Theterm encompasses a “nucleic acid profile” that analyzes one or moregenetic markers, a “protein profile” that analyzes one or morebiochemical or serological markers, and combinations thereof. Examplesof nucleic acid profiles include, but are not limited to, a genotypicprofile, gene copy number profile, gene expression profile, DNAmethylation profile, and combinations thereof. Non-limiting examples ofprotein profiles include a protein expression profile, proteinactivation profile, and combinations thereof. For example, a “genotypicprofile” includes a set of genotypic data that represents the genotypeof one or more genes associated with a tumor, tumor cell, and/or cancer.Similarly, a “gene copy number profile” includes a set of gene copynumber data that represents the amplification of one or more genesassociated with a tumor, tumor cell, and/or cancer. Likewise, a “geneexpression profile” includes a set of gene expression data thatrepresents the mRNA levels of one or more genes associated with a tumor,tumor cell, and/or cancer. In addition, a “DNA methylation profile”includes a set of methylation data that represents the DNA methylationlevels (e.g., methylation status) of one or more genes associated with atumor, tumor cell, and/or cancer. Furthermore, a “protein expressionprofile” includes a set of protein expression data that represents thelevels of one or more proteins associated with a tumor, tumor cell,and/or cancer. Moreover, a “protein activation profile” includes a setof data that represents the activation (e.g., phosphorylation status) ofone or more proteins associated with a tumor, tumor cell, and/orcancer.”

The terms “level” and/or “activity” as used herein further refer to geneand protein expression levels or gene or protein activity. For example,gene expression can be defined as the utilization of the informationcontained in a gene by transcription and translation leading to theproduction of a gene product.

In certain non-limiting embodiments, an increase or a decrease in asubject or test sample of the level of measured biomarkers (e.g.proteins or gene expression) as compared to a comparable level ofmeasured proteins or gene expression in a control subject or sample canbe an increase or decrease in the magnitude of approximately±5,000-10,000%, or approximately ±2,500-5,000%, or approximately±1,000-2,500%, or approximately ±500-1,000%, or approximately ±250-500%,or approximately ±100-250%, or approximately ±50-100%, or approximately±25-50%, or approximately ±10-25%, or approximately ±10-20%, orapproximately ±10-15%, or approximately ±5-10%, or approximately 1-5%,or approximately ±0.5-1%, or approximately ±0.1-0.5%, or approximately±0.01-0.1%, or approximately ±0.001-0.01%, or approximately±0.0001-0.001%.

The values obtained from controls are reference values representing aknown health status and the values obtained from test samples orsubjects are reference values representing a known disease status. Theterm “control”, as used herein, can mean a sample of preferably the samesource (e.g. blood, serum, tissue etc.) which is obtained from at leastone healthy subject to be compared to the sample to be analyzed. Inorder to receive comparable results the control as well as the sampleshould be obtained, handled and treated in the same way. In certainexamples, the number of healthy individuals used to obtain a controlvalue may be at least one, preferably at least two, more preferably atleast five, most preferably at least ten, in particular at least twenty.However, the values may also be obtained from at least one hundred, onethousand or ten thousand individuals.

A level and/or an activity and/or expression of a translation product ofa gene and/or of a fragment, or derivative, or variant of saidtranslation product, and/or the level or activity of said translationproduct, and/or of a fragment, or derivative, or variant thereof, can bedetected using an immunoassay, an activity assay, and/or a bindingassay. These assays can measure the amount of binding between saidprotein molecule and an anti-protein antibody by the use of enzymatic,chromodynamic, radioactive, magnetic, or luminescent labels which areattached to either the anti-protein antibody or a secondary antibodywhich binds the anti-protein antibody. In addition, other high affinityligands may be used. Immunoassays which can be used include e.g. ELISAs,Western blots and other techniques known to those of ordinary skill inthe art (see Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999 andEdwards R, Immunodiagnostics: A Practical Approach, Oxford UniversityPress, Oxford; England, 1999). All these detection techniques may alsobe employed in the format of microarrays, protein-arrays, antibodymicroarrays, tissue microarrays, electronic biochip or protein-chipbased technologies (see Schena M., Microarray Biochip Technology, EatonPublishing, Natick, Mass., 2000).

Certain diagnostic and screening methods of the present inventionutilize an antibody, preferably, a monocolonal antibody, capable ofspecifically binding to a protein as described herein or activefragments thereof. The method of utilizing an antibody to measure thelevels of protein allows for non-invasive diagnosis of the pathologicalstates of kidney diseases. In a preferred embodiment of the presentinvention, the antibody is human or is humanized. The preferredantibodies may be used, for example, in standard radioimmunoassays orenzyme-linked immunosorbent assays or other assays which utilizeantibodies for measurement of levels of protein in sample. In aparticular embodiment, the antibodies of the present invention are usedto detect and to measure the levels of protein present in a renal cellor urine sample.

Humanized antibodies are antibodies, or antibody fragments, that havethe same binding specificity as a parent antibody, (i.e., typically ofmouse origin) and increased human characteristics. Humanized antibodiesmay be obtained, for example, by chain shuffling or by using phagedisplay technology. For example, a polypeptide comprising a heavy orlight chain variable domain of a non-human antibody specific for adisease related protein is combined with a repertoire of humancomplementary (light or heavy) chain variable domains. Hybrid pairingsspecific for the antigen of interest are selected. Human chains from theselected pairings may then be combined with a repertoire of humancomplementary variable domains (heavy or light) and humanized antibodypolypeptide dimers can be selected for binding specificity for anantigen. Techniques described for generation of humanized antibodiesthat can be used in the method of the present invention are disclosedin, for example, U.S. Pat. Nos. 5,565,332; 5,585,089; 5,694,761; and5,693,762. Furthermore, techniques described for the production of humanantibodies in transgenic mice are described in, for example, U.S. Pat.Nos. 5,545,806 and 5,569,825.

In order to identify small molecules and other agents useful in thepresent methods for treating or preventing a renal disorder bymodulating the activity and expression of a disease-related protein andbiologically active fragments thereof can be used for screeningtherapeutic compounds in any of a variety of screening techniques.Fragments employed in such screening tests may be free in solution,affixed to a solid support, borne on a cell surface, or locatedintracellularly. The blocking or reduction of biological activity or theformation of binding complexes between the disease-related protein andthe agent being tested can be measured by methods available in the art.

Other techniques for drug screening which provide for a high throughputscreening of compounds having suitable binding affinity to a protein, orto another target polypeptide useful in modulating, regulating, orinhibiting the expression and/or activity of a disease, are known in theart. For example, microarrays carrying test compounds can be prepared,used, and analyzed using methods available in the art. See, e.g.,Shalon, D. et al., 1995, International Publication No. WO95/35505,Baldeschweiler et al., 1995, International Publication No. WO95/251116;Brennan et al., 1995, U.S. Pat. No. 5,474,796; Heller et al., 1997, U.S.Pat. No. 5,605,662.

Identifying small molecules that modulate protein activity can also beconducted by various other screening techniques, which can also serve toidentify antibodies and other compounds that interact with proteinsidentified herein and can be used as drugs and therapeutics in thepresent methods. See, e.g., Enna et al., eds., 1998, Current Protocolsin Pharmacology, John Wiley & Sons, Inc., New York N.Y. Assays willtypically provide for detectable signals associated with the binding ofthe compound to a protein or cellular target. Binding can be detectedby, for example, fluorophores, enzyme conjugates, and other detectablelabels well known in the art. The results may be qualitative orquantitative.

For screening the compounds for specific binding, various immunoassaysmay be employed for detecting, for example, human or primate antibodiesbound to the cells. Thus, one may use labeled anti-hIg, e.g., anti-hIgM,hIgG or combinations thereof to detect specifically bound humanantibody. Various labels can be used such as radioisotopes, enzymes,fluorescers, chemiluminescers, particles, etc. There are numerouscommercially available kits providing labeled anti-hIg, which may beemployed in accordance with the manufacturer's protocol.

By way of a mechanism of the present invention and without being limitedby way of theory, the inventors of the present application postulatethat in certain malignancies, the apoptotic escape can be initiated atthe level of the second messenger (3′-5′-cyclic adenosine monophosphate(cAMP), for example). The tumor cells develop the mechanism that removesthe second messenger from the cytoplasm, and as a consequence, promotesan apoptotic escape, resulting in malignancies. The present invention isbased upon the concept that it is possible to reprogram cancer cells byblocking the second messenger removal using drugs or drug-likecompounds, which are presented herein. This approach can be applied tothe treatment of malignancies on an individualized or patient by patientbasis, where second messenger removal represents a part of ananti-apoptotic pathway. Methods of inhibiting, reducing the likelihoodor treating cancer, especially including metastatic cancer are importantembodiments of the present invention.

Pursuant to the present invention, the inventors propose the followingmechanisms for the prevention and/or treating of cancer, includingmetastatic cancer using compounds according to the present invention:

-   -   The inventors propose that accumulation of cAMP represents part        of the normal mechanism responsible for the elimination of        damaged, precancerous cells.    -   The inventors further propose that the mechanisms of apoptotic        escape, employed by a cancer cell, can be related to a decrease        in the cytoplasmic cAMP concentration. This can be achieved by        removing cAMP from the cytoplasm by transporters.    -   The inventors further propose that targeting cAMP efflux using        specific blockers as described herein in certain malignancies        will overcome apoptotic escape, and thus, provide a novel        therapeutic option, particularly effective when combined with        additional anticancer agents, which exhibit biological activity        separate from the restoration of apoptotic mechanism by        compounds according to the present invention.    -   The inventors also propose that cAMP efflux can be targeted on        an individualized (patient by patient) basis with respect to the        overexpression of efflux activity, as well as the reprogramming        of phenotypic responses including viability, proliferation,        apoptosis, cell cycle, and phosphoprotein levels.    -   The inventors propose that the use of F-cAMP may represent a        diagnostic biomarker to predict the responsive of patient cells        ex vivo and that the loss of F-cAMP efflux from patient samples        may represent a measure of the effectiveness of the proposed        treatment.    -   The inventors propose that a reduction of cAMP in the urine of        patients treated with the proposed compounds may represent a        measure of the effectiveness of the proposed treatment.

These and other aspects of the invention are illustrated further in thefollowing non-limiting examples.

Example 1 Effect of cAMP Efflux Blockers

To test the basic hypothesis, the inventors developed an assay for thedetection of the efflux of the cAMP fluorescent analog, and screened thePrestwick Chemical Library and the SPECTRUM Collection for blockers ofcAMP efflux. These libraries are composed of FDA approved drugs anddrug-like compounds, and therefore, represent a valuable source for drugrepurposing. We identified:

-   -   1) A number of structurally related compounds that blocked        cAMP-analog efflux in a dose-dependent manner.    -   2) In secondary assays these compounds when used alone rapidly        decreased cell viability, induced cell cycle arrest, and        promoted apoptosis. EC50s for cell cycle arrest and apoptosis        were comparable with IC50s for cAMP efflux.    -   3) The normal (non-malignant) human peripheral blood cells were        less sensitive to the second messenger blockers, suggesting that        removal of the second messenger from the cytoplasm represents a        cancer cell-specific mechanism.        Results        Assay for the Detection of Cyclic AMP Efflux

To study the efflux of cAMP from cells we used the Alexa Fluor 488conjugated cAMP analog (FIG. 2, hereof), commercially available fromLife Technologies. U937 cell were loaded with the analog by a methodrelying on osmotic lysis of pinocytic vesicles (15). After incubation,cell fluorescence decreased to a level that was close to cellautofluorescence. MK-571 is a compound reported to block cAMP efflux ina dose-dependent manner.

High Throughput Flow Cytometry Screen

Using the above-described assay, the inventors screened the PrestwickChemical Library (Prestwick Chemical) and the SPECTRUM Collection(Microsource Discovery Systems, Inc.) at the University of New MexicoCenter for Molecular Discovery (UNMCMD, http://nmmlsc.health.unm.edu/).We have identified four commercially available drugs and severalstructurally related compounds that blocked cAMP-analog efflux in adose-dependent manner.

According to the inventors' hypothesis, blocking cAMP efflux shouldresult in the loss of cell viability, cell cycle arrest, and apoptosis.To test this, the inventors performed a number of secondary assays.

Secondary Assays: Cell Viability, Cell Cycle, Proliferation, andApoptosis

Cell viability. The CellTiter-Glo® luminescent cell viability assay isbased on quantification of intracellular ATP and serves as an indicatorof metabolically active “viable” cells. Overnight incubation withdifferent concentrations of the screening hits showed a significantdecrease in cell viability (FIG. 2). For several compounds, the IC50swere significantly lower as compared to the MK-571 (control). Thus, anumber of compounds identified as cAMP efflux blockers were able torapidly decrease cellular ATP content.

Cell cycle. The inventors used propidium iodide for DNA staining todetect the phases of the cell cycle. Several identified compoundsinduced cell cycle arrest in the G0/G1 phase, decreasing the percentageof cells in G2/M and S phases in a dose-dependent manner. G1 arrest inmonocytes is reported to be controlled by a cAMP/p27/Kip1-relatedmechanism (8). Seven compounds also exhibited a dose dependent increasein the number of cells with DNA content less than the G0/G1 phase(dead/apoptotic cells). Thus, selected compounds identified as cAMPefflux blockers were able to induce cell cycle arrest/apoptosis whenused alone in a dose-dependent manner.

Cell Proliferation. To assess cell proliferation, the inventors used theCellTrace™ CFSE Cell Proliferation Kit. Cells were stained withcarboxyfluorescein diacetate succinimidyl ester (CFSE). This dye formsdye-protein adducts that are retained during cell division, and thefluorescent dye was used to quantify cell proliferation. We found that anumber of compounds when used at 3-6 μM concentration stopped cellproliferation 48 hours after compound addition. These data wereconsistent with the results of the cell cycle assay.

Apoptosis.

To detect apoptosis we used an Annexin V and 7-amino-actinomycin D(7-AAD) based assay. Annexin V is a protein that has high bindingaffinity to phosphatidylserine, which is translocated from the inner tothe outer leaflet of the membrane during early apoptosis. 7-AAD is a DNAdye that stains cells only when the cell membranes are permeable. Thus,double positive events are considered to represent end stage apoptosisand dead cells. This assay does not discriminate between cells that haveundergone apoptotic death versus a necrotic pathway. Our data indicatedthat a number of compounds identified as blockers of cAMP efflux inducedrapid dose-dependent cell death (FIG. 4). The results obtained usingthis assay correlated well with the results of other assays:

Calculated EC₅₀ values from Annexin V, 7-AAD double positive events, μMArtemisinin 248.89 Artemether 106.41 Artesunate 99.08 Dihydroartemisinin55.72 Patulin 3.29 Pyrithione zinc 11.22 Parthenolide 13.52 Quinalizarin172.19 Clioquinol 76.56 Cryptotanshinone 53.46 Harmalol 283.14

See also FIG. 4A

Thus, secondary assays confirmed that selected cAMP efflux blockers,when used alone: 1) rapidly induced a decrease in cell viability; 2)induced cell cycle arrest in G0/G1 phase, as consistent with the effectof cAMP(8); 3) rapidly stopped cell proliferation; and 4) inducedapoptosis/cell death as measured by Annexin V and 7-Aminoactinomycin D,and DNA fragmentation. EC50s for cell cycle arrest and apoptosis werecomparable with IC50s for cAMP efflux. None of the identified compoundswere chemotherapeutic drugs.

Another prediction that can be made according to the present inventionis that cells that do not possess the cAMP efflux mechanism (for examplenormal non-cancerous cells) should exhibit less sensitivity to cAMPblockers. This is postulated because normal cells do not require cAMPremoval to sustain cell survival. To test this prediction the presentinventors compared the effect of cAMP efflux blockers on the viabilityof peripheral blood mononuclear cells (PBMCs) side by side with U937cells.

Effect of cAMP Efflux Blockers on Human PBMCs

The viability curves for human PBMCs, treated with the array ofidentified compounds showed a significant shift to the right as comparedto U937 cells (FIG. 5). This indicates that normal peripheral bloodcells were less sensitive to the effect of cAMP blockers.

FIG. 5A shows the effects of the identified cAMP efflux inhibitors onU937 proliferation. Cells were stained with CFSE in bulk, and thenseparated into cultures containing either solvent, 3 μM, or 10 μMcompounds. Samples were taken from cultures after 48, 72, and 96 hoursafter initial culture time. A and B) Raw data of CFSE MFI in cellsremaining after treatment with hit compounds, as measured by flowcytometry. Decreases in CFSE MFI over time indicate cell proliferation.Data in (B) were analyzed by one-way ANOVA with repeated measures with aDunnett post test to compare treated samples to DMSO control values(n=3, *p<0.05, **p<0.01, ***p<0.001). Data shown are the result of 3independent experiments.

Additionally, the inventors tested the ability of human PBMCs to effluxthe Alexa488-cAMP analog in the primary assay, as shown in FIG. 2. Theresults suggest that U937 cells have a greater ability to removeAlexa488-cAMP analog from the cytoplasm than PBMCs.

Thus, a number of compounds (FIG. 1) identified as blockers of cAMPefflux when used alone stopped cell proliferation, induced the loss ofcell viability and cell apoptosis. Normal human peripheral bloodmononuclear cells where less sensitive to the effects of the compoundspresumably because of the lack of the cAMP removal system that isunnecessary for normal (non-cancerous cell survival).

Example 2 Assessing cAMP-Concentrations after Treatment with Drugs

To test our central hypothesis, we developed a novel assay, and screenedthe Prestwick Chemical Library (Prestwick Chemical) and the SPECTRUMCollection (Microsource Discovery Systems, Inc.) at the University ofNew Mexico Center for Molecular Discovery. We found that fourcommercially available drugs and several structurally related compoundsblocked cAMP efflux in a dose dependent manner. Secondary assaysconfirmed that the compounds identified, when used alone: 1) inducedcell cycle arrest in the G1 phase, as consistent with the effect ofcAMP; 2) induced apoptosis as measured by Annexin V and7-Aminoactinomycin D, and DNA fragmentation; 3) rapidly induced adecrease in cell viability. EC50s for cell cycle arrest and apoptosiswere comparable with IC50s for the cAMP efflux. Thus, our data provide aproof of concept, and support the central hypothesis. These data havebeen disclosed as a patent application (“Method for Cancer CellReprogramming” (STC ref. 2013-097)).

Next, because several types of genetic rearrangements contribute tocancer cell phenotypes, we studied heterogeneity of cAMP removal systemsin a set of genetically diverse cell lines. Two cell lines were shown tohave identical genetic rearrangement and a fusion gene (Table 1) (21).The set of proof of principle data disclosed in the patent applicationwas obtained using U937 cell line (AML, t(10; 11)(p12;q14),PICALM/MLLT10(AF10)).

TABLE 1 The human cell lines included in the study, their subtype andgenetic rearrangements (21) Genetic Cell line Subtype rearrangementFusion gene 697 (EU-3) B-lineage ALL t(1; 19)(q23; p13) TCF3(E2A)/PBX1¹Nalm-6 B-lineage ALL t(5; 12)(q33; p13) Sup-B15 B-lineage ALL t(9;22)(q34; q11) P190 BCR/ABL1 REH B-lineage ALL t(12; 21)(q13; q22)ETV6(TEL)/ RUNX1(AML1) RS4:11 B-lineage ALL t(4; 11)(q21; q23)MLL/MLLT2(AF4) Mhh-Call 3 B-lineage ALL t(1; 19)(q23; p13)TCF3(E2A)/PBX1¹ ¹Two cell lines have identical genetic rearrangement anda fusion gene.

To study the differences in cAMP-removal, cells were loaded with afluorescent cAMP-analog, and incubated under different conditions: lowtemperature (4° C.) used to estimate the passive probe “leakage”, andphysiological temperature (37° C.) used to assess active removal of theprobe. MK-571, a previously reported inhibitor of cAMP efflux, was usedto estimate the “pump-dependent” component of the process (19).

The cAMP-removal system in cancer cell lines exhibited dramaticallydiverse behavior (FIG. 6). Because cells loaded with the fluorescentprobe were subsequently washed, the resulting concentration gradient cancause a “passive leak” of the probe. After incubation at 4° C., celllines have lost 20-60% of the initial stain. Incubation at 37° C.resulted in removal of the probe from ˜60% for SUP-B15 and MHH-CALL3cells, down to ˜90% for 697 and Nalm-6 cells with no apparentcorrelation between 4° C. and 37° C. samples. Cell lines also exhibiteddifferent sensitivity to the effect of MK-571, a known cAMP-blocker. Theresponse ranged from 0-60%, where “0” corresponds to the absence ofefflux. For two cell lines, SUP-B15, and MHH-CALL3, the inhibitorcompletely blocked cAMP probe efflux. For these cell lines MK-571blocked not only active probe removal at 37° C., but also efflux at 4°C., and therefore the fluorescent signal was equal to the initial cAMPprobe loading value (FIG. 6). Thus, the cAMP removal system exhibited alarge degree of variability. Moreover, these effects were not directlyrelated to the type of genetic rearrangement. For the two lines (697 andMhh-Call3) with an analogous fusion protein (Table 1), the cAMP-removalwas drastically different (FIG. 6). This suggests that thegenomics/sequencing approaches will not be sufficient for cAMP-effluxprediction.

Finally, to establish whether the effects of identified cAMP-effluxblocker compounds on cell viability can be directly linked to theaccumulation of cAMP, we studied the dose responses for cAMPaccumulation and cell viability (FIG. 7). We observed a correlationbetween cAMP accumulation and a decrease in the cell viability for sevenmolecules that were identified in a screen as inhibitors of cAMP-efflux.Quantitatively, the coefficient of determination (r²) for thecorrelation between EC50s for the drug-induced loss of cell viabilityplotted versus EC50s for the drug-induced accumulation of cAMP in thecytoplasm was ˜0.98 (FIG. 2C), suggesting a very strong correlation.

Taken together, these data support our prediction that assessingcAMP-concentrations after treatment with drugs can serve as a predictorfor the efficacy of a particular drug in patient samples. Patientsamples will be analyzed in a manner identical to the cell lines (asdetailed in Research design and Methods), and samples exhibiting thehighest sensitivity to cAMP blocking therapy will be validated forfurther therapeutic options.

Example 3 Identification of Cyclic AMP Efflux Inhibitors as PotentialTherapeutic Agents for Hematological Malignancies

Here, we used a fluorescent cAMP analog (F-cAMP) in a flow cytometricassay to monitor cAMP efflux in leukemic cells. Next, to identifycompounds and drugs that could inhibit cAMP efflux, we miniaturized theassay into a high throughput screening (HTS) format and screened twosmall molecule libraries composed of biologically active substances andoff-patent drugs in U937 acute myeloid leukemia (AML) cells. The “hits”were validated by secondary assays, which assessed the effect of thecompounds on viability, proliferation, cell cycle, and apoptosis. Next,these compounds were tested for effects on cAMP efflux inhibition andviability in B-lineage acute lymphoblastic leukemia (ALL) cells, an AMLpatient sample, and healthy human primary blood mononuclear cells(PBMCs). Our hypothesis was further supported by measurements ofendogenous cAMP accumulation in B-lineage ALL cells after exposure tothe hit compounds. Because several of the compounds identified are FDAapproved drugs, our studies provide a path for clinical trials of thesecompounds for drug repurposing [repositioning] against blood cancers.

Materials and Methods

Cells

U937 cells were obtained from ATCC. They were grown in RPMI 1640 medium(supplemented with 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mLstreptomycin, 10 mM HEPES, and 10% heat-inactivated fetal bovine serum(FBS), hereafter referred to as cRPMI), and kept at 37° C. and 5%CO₂/95% air.

cAMP efflux assay

This method of loading U937 cells with a fluorescent analog of cAMP isbased on a procedure described by Okada, et al. 1982¹⁹. Briefly, cellswere concentrated and washed, with resuspension NF-RPMI (cRPMI withoutFBS). Cells were then resuspended in an NF-RPMI hypertonic solutioncontaining poly(ethylene glycol) 1,000 (PEG), sucrose, and Alexa Fluor®488 8-(6-aminohexyl) aminoadenosine 3′,5′-cyclicmonophosphate,bis(triethylammonium) salt, hereafter referred to as F-cAMP, to give afinal concentration of 4.76 mM F-cAMP. Cells were incubated for 10 min,room temperature, then centrifuged and resuspended in hypotonic solution(60% cRPMI, 40% sterile water) for 2 min at room temperature to completethe F-cAMP loading. The cells were then washed and resuspended in cRPMIat a final concentration of 4×10⁵ cells/mL, and allowed to equilibratefor 2 hours in a 37° C., 5% CO₂/95% air incubator.

For general testing of cAMP efflux, a small sample of the stained cellswas retained and kept at 4° C. overnight to serve as a control. Theremainder of the cells was incubated (37° C., 5% CO₂/95% air) in thepresence of dimethyl sulfoxide (DMSO) vehicle or compounds overnight.Flow cytometry was used to measure samples, and unstained U937 cellswere used to create a gate to mark viable cells. Control andexperimental samples were excited with a 488 nm laser and analyzed forFL-1 median fluorescence intensity (MFI) within that gate.

High Throughput Screening

For the high throughput screening assay (HTS), U937 cells were loadedwith F-cAMP as described above. Solutions were added to 384-well plates(Greiner 784201) with a Biomek FX Multichannel system (Beckman-Coulter)and/or MicroFlo as follows: 1) 5 μL cRPMI; 2) 100 nL compounds from thePrestwick Chemical Library or Spectrum 2000 Library in DMSO weredelivered by pintool (V&P Scientific, San Diego, Calif.), final DMSOconcentration=1%; 3) 5 μL F-cAMP cells. Negative control wells containedstained cells with 1% DMSO only. For positive controls, F-cAMP cellswere treated with 200 μM MK-571. The final concentration for allconditions was 2000 cells/well. The plates were sealed with foil, andincubated (37° C., 5% CO₂/95% air) inverted overnight.

After incubation, the 384-well sample plates were analyzed by CyAn flowcytometers (Beckman-Coulter) configured with HyperCyt high throughputauto-sampler systems (IntelliCyt Albuquerque, N. Mex.). The samples wereinterrogated with 488 nm lasers to assess FL-1 MCI levels.

HTS Data Analysis and Hit Compound Validation

Data from the HTS were analyzed with HyperView software (IntelliCyt,Albuquerque, N. Mex.) and gated on untreated, live cell populations andthen time-gated to separate data per well. FL-1 MCI values for thesamples were analyzed per plate. Those samples which reported MCIvalues≥2 standard deviations above the plate mean negative controlvalues were considered “hit” compounds.

To validate the identified samples and decrease the number of potentialfalse-positive compounds, the hit compounds were assayed in ahigh-throughput dose response assay. This assay set up 384-well plateswith the same volumes of F-cAMP cells, cRPMI, and DMSO/compounds as inthe HTS, with the plate formats containing 10-well dose responses foreach hit compound, at final concentrations ranging 30 μM to 4 nM. Theseplates were also foil-sealed, and incubated inverted overnight.Post-incubation, the dose response plates were analyzed by highthroughput flow cytometry, as above.

The dose response data was fitted by Prism software (GraphPad Software,Inc., La Jolla, Calif.) and normalized for percent response based onsample FL-1 MCI values in comparison to untreated control F-cAMP-loadedcells kept at 4° C. overnight. These analyses lead to the identificationof 8 compounds with clear dose response curves and decent EC₅₀ values.An additional 3 compounds were identified for testing based onstructural relatedness to these 8 key compounds.

Secondary Assays

Apoptosis:

Six-well plates were set up with dose responses for each of the 11 hitcompounds (artemisinin, artemether, artesunate, dihydroartemisinin,parthenolide, patulin, clioquinol, cryptotanshinone, pyrithione zinc,harmalol, quinalizarin). Each well contained 2.5×10⁶ U937 cells in 5 mLcRPMI. To each well, 15 μL of compound in DMSO (or vehicle control) wasfTadded, giving final concentrations of 0, 300 nM, and 1, 3, 10, and 30Plates were incubated overnight, at 37° C., 5% CO₂/95% air. The compoundeffects on apoptosis were assessed with an AnnexinV-PE/7-amino-actinomycin D (7-AAD) kit (BD Pharmingen™ cat. no. 559763),according to manufacturer protocol, and data were collected with a BDAccuri™ C6 flow cytometer. A gate was set for live cells, and 10,000gated events were collected per sample. FL-2 versus FL-3 channel dotplots were divided into quadrants and were used to determine percentagesof gated cells that were Annexin V⁻/7-AAD⁻ (live, healthy cells),Annexin V⁺/7-AAD⁻ (early apoptosis), or Annexin V⁺/7-AAD⁺ (late, fullapoptosis).

Cell Cycle:

The initial steps of the cell cycle assay were set up with the same U937cell density, volume, compound addition, culture conditions, andincubation as described for the apoptosis assay above. After 24 hourincubation, the samples were centrifuged and fixed in 5 mL 70% ethanolat 4° C. for at least one week. After fixation, samples were washed withPBS and stained with propidium iodide (PI) staining solution (0.1% v/vTriton X-100, 10 μg/mL PI, 100 μg/mL DNase-free RNase A in PBS) for 30min at room temperature, in the dark. Samples were then interrogatedwith a BD Accuri™ C6 flow cytometer, and FL-2 channel histograms weregated to determine percentages of cells that were in apoptotic (<2nDNA), G₀/G₁ (2n DNA), S (2n<DNA<4n), or G₂/M (4n DNA) phases of the cellcycle.

Proliferation:

To assess cell proliferation in the presence of compounds, theCellTrace™ CFSE (carboxyfluorescein diacetate succinimidyl ester) CellProliferation Kit (Molecular Probes®) was used. To incorporate CFSE intoU937 cells, 10⁶/mL cells were resuspended in 25 μM CFSE in PBS, andincubated 15 min in a 37° C. water bath. The cells were centrifuged,resuspended in cRPMI at 10⁵ cells/mL, and incubated for 30 min in a 37°C. water bath. The cells were then washed once and resuspended in cRPMIat 10⁴ cells/mL. These CFSE-labeled U937 cells were cultured as follows:5 mL cells/well in a 6-well tissue culture plate. Each 6-well platecontained a DMSO-only negative/0 μM control. For this assay, onlyartesunate, dihydroartemisinin, patulin, pyrithione zinc,cryptotanshinone, and parthenolide were tested, at final concentrationsof 3 and 10 μM. All plates were kept in a 37° C., 5% CO₂/95% airincubator. A small aliquot of freshly-labeled cells was retained andanalyzed with a FACScan flow cytometer to determine the MFI for theinitial staining. At 48 hr post-CFSE staining, 500 μL volumes wereobtained from each sample and analyzed. This was repeated at 72 and 96hr time points. CFSE MFI values were analyzed by one-way ANOVA withrepeated measures, with a Dunnett post test to compare treated samplesto DMSO control values.

Viability:

Viability of cells in the presence of hit compounds was determined withthe Cell TiterGlo® Luminescent Cell Viability Assay (Promega). Opaque,white 96-well tissue culture plates (Greiner Bio-One 655083) received100 μL medium (cRPMI or 20% FBS cRPMI in the case of MHH Call 3cells)+/−4×10⁴ cells/well. Dose responses were 1:4 dilutions of 1 μL ofcompounds which ranged from final concentrations of 100 μM to 1.53 nM(final DMSO concentration=1%). Wells with DMSO only served as negativecontrols, while wells with final concentrations of 500 μM daunorubicinserved as positive controls. Cells were incubated overnight under normaltissue culture conditions, in 37° C., 5% CO₂/95% air incubators. Afterincubation, the Promega Cell TiterGlo® Luminescent Cell Viability Assaywas performed according to manufacturer's protocol.

PBMCs:

Healthy human primary blood mononuclear cells (PBMCs) were obtained fromvolunteers. PBMCs were treated with the same compounds in dose response,and culture conditions, as in the viability assay described above.

Cross-Cell Line Tests for cAMP Efflux:

These assays followed the methods described for the original cAMP effluxassay for the following B-lineage ALL cell lines: 697, Reh, MHH Call 3,RS4; 11, Sup B15, and Nalm 6. Briefly, 15 million cells were loaded withF-cAMP via the osmotic process detailed above. Small volumes of cellswere analyzed by flow cytometry post-staining to determine baselinefluorescence values based on MFI. Additional small quantities of cellswere kept at 4° C. overnight to serve as controls for passive leakage ofcAMP. The remaining cells were split in half, and one group was treatedwith a final concentration of 100 μM MK-571, while the other receivedDMSO-only at an equal volume. These two groups of cells were incubated˜24 hr according to standard tissue culture conditions (37° C., 5%CO₂/95% air). After incubation, cells were analyzed with flow cytometryfor FL-1 MFI.

Cross-cell line high throughput tests of hit compound inhibition of cAMPefflux:

This assay was conducted the same as the high throughput hit validationassay described above, with the exception that cell densities were 5,000per well. Briefly, each B-lineage ALL cell line (697, Reh, MHH Call 3,RS4; 11, Sup B15, or Nalm 6) was incorporated with F-cAMP, and cellswere loaded into 384-well plates in the presence of the top 8 hitcompounds, in dose response, with final concentrations ranging in 1:4dilutions from 100 μM to 1.53 nM. Plates were sealed with foil andincubated inverted overnight (37° C., 5% CO₂/95% air). Post-incubation,plates were vortexed and analyzed by high throughput flow cytometry forresidual F-cAMP via FL-1 MFI.

Cross-Cell Line Tests for Viability with Hit Compounds:

The B-lineage ALL cell lines 697, Reh, MHH Call 3, RS4; 11, Sup B15, andNalm 6, were run through the Cell TiterGlo® Luminescent Cell ViabilityAssay with the same compounds, cell densities, and conditions asdescribed above. The top 8 hit compounds were tested: artesunate,dihydroartemisinin, parthenolide, patulin, MK-571, pyrithione zinc,clioquinol, and cryptotanshinone. The same was repeated with a frozenpatient AML sample (with the exception that the cell density used was3,000 cells per well.

Cross-Cell Line Analysis for Presence of MRP4 (ABCC4):

Two million cells each of 697, Reh, MHH Call 3, RS4; 11, Sup B15, andNalm 6 cell lines were fixed with 80% methanol/diH₂O for 5 min at roomtemperature. The cells were centrifuged and resuspended in 1 mL 0.3Mglycine in a 10% FBS in PBS solution. The samples were divided into 500μL portions, one each for MRP4 antibody labeling, and IgG isotypecontrol. MRP4 samples were labeled with 1 μg/μL primary MRP4 antibodies(goat, anti-human). Control samples were labeled with 2 μg/μL primaryIgG antibodies (goat, anti-human). All primary antibody-labeled sampleswere incubated for 30 min at room temperature, then washed once withPBS, and resuspended in 500 μL 0.3M glycine in 10% FBS in PBS. Then, 1μL of FITC×anti-goat secondary antibody was added to all samples,followed by 30 min room temperature incubation. Additionally, one samplewas made consisting of a set of Quantum™ Simply Cellular® calibrationbeads (Bangs Laboratories, Inc.), and this was subjected to the sameprotocol as with the MRP4 samples. Post-labeling, all samples were runon a BD Accuri™ C6 flow cytometer with 488 nm laser excitation foranalysis of FITC (FL-1) MFI. The calibration beads were gatedindividually, and the median channel fluorescence of these was plottedagainst known antibody binding complexes (ABCs) per bead, and a linearregression was applied. The derived equation was used to calculate thenumber of ABCs per sample. Then, for each cell type, the ABC value forIgG isotype control samples was subtracted from that of the MRP4 samplesto determine the number of MRP4-specific binding sites.

cAMP Accumulation in the Presence of Hit Compounds:

This method utilized the Promega cAMP-Glo™ assay kit, and the cell linesU937, 697, Reh, MHH Call 3, RS4; 11, Sup B15, and Nalm 6 were tested,along with a sample of healthy PBMCs. Cells were centrifuged 10 min andresuspended in cRPMI supplemented with 500 μM3-isobutyl-1-methylxanthine (IBMX), 100 μM 4-(3-butoxy-4-methoxy-benzyl)imidazolidone (Ro 20-1724), and 25 mM MgCl₂ (cRPMI-IRM) at a density of2.5×10⁶/mL. The subsequent setup of these plates was the same asoccurred in the high throughput cAMP efflux inhibition assay describedabove, with the exception that there were 12,500 cells/well, and 10 μMforskolin served as positive control. After overnight incubation, 4 μL(5,000 cells) from each well was transferred to opaque white shallow384-well plates (Greiner Bio-One 784080) for the completion of thecAMP-Glo™ assay. The remainder of the assay was followed according tothe manufacturer's protocol, including the usage of a separate assayplate containing known concentrations of cAMP in dose response togenerate a standard curve. Briefly, 1 μL of cAMP Detection Solution wasadded to each well and incubated 20 min at room temperature. Then, 5 μLKinase-Glo® reagent was added, and plates were incubated a minimum of 10min at room temperature. A plate reader was used to measure relativeluminescence units (RLU) of each well at 1 sec/well. To analyze data,the ΔRLU ((RLU 0 nM cAMP)-(RLU (X nM)) for the standard cAMPconcentrations was generated, plotted, and fit with a linear regressionanalysis. Then, for assay samples, the ΔRLU (RLU (neg. control)-RLU(sample) was calculated and utilized in the linear equation derived fromthe cAMP standard curve to determine cAMP concentration.

Results

A Fluorescent cAMP Analog can be Used to Measure cAMP Efflux with a HighThroughput Screen for Inhibitors by Using Flow Cytometry

Earlier studies in U937 cells, an established model for AML, had shownthat these cells efflux cAMP. Therefore, we sought to create an assay toassess cAMP efflux in U937 cells. This method is derivative of Okada,1982¹⁹, in which large molecules were incorporated into cells viainduced osmotic lysis of pinocytic vesicles. A fluorescent cAMP analog,Alexa 488-conjugated cAMP (F-cAMP), was loaded into cells, and MFI wasassessed after overnight incubation under normal conditions; a samplekept at 4° C. served as a high fluorescence control (data not shown).This strategy is based on the premise that active removal of F-cAMP willcause a decrease in the signal, while those cells retaining F-cAMP wouldmaintain green fluorescence comparable to the control. It has been notedthat the compound MK-571 is known to inhibit cAMP efflux¹¹, and itseffectiveness was tested in this assay by adding the compound to cells afew hours after they were loaded with F-cAMP. As shown in FIG. 2, MK-571down-modulated cAMP efflux in a dose-dependent manner.

These preliminary experiments served as the basis for development of ahigh throughput screening (HTS) assay to identify drugs and compoundswhich may potentially block cAMP efflux in leukemic cells. The assay wasminiaturized and optimized to work at small volume in 384-well plates.Briefly, bulk amounts of cells were loaded with F-cAMP and allowed toequilibrate for a few hours. Medium, F-cAMP-loaded cells, and testcompounds from the Prestwick Chemical Library (˜1200 previouslyFDA-approved drugs) and the SPECTRUM Collection (2320 compounds-60%drugs, 25% natural products, 15% bioactive components) were added towells in 384-well plates and incubated overnight. DMSO served as anegative control, while 100 μM MK-571, well above the 31 μM EC₅₀calculated in assay development, was the positive control.

Potential “hit” compounds were identified as having MFI's>2 standarddeviations above the negative control MFI. This analysis identified 51hits, and these were tested in dose response to validate theiractivities in the assay (data not shown). These dose responses yielded 8potentially useful molecules.

The Identified cAMP Efflux Inhibitors Increased Cancer Cell Apoptosis

The effect of cAMP efflux inhibitors on U937 apoptosis was detected viaflow cytometry with the Annexin V-PE/7-amino-actinomycin D (7-AAD) assayto discriminate dead and dying cells. Samples were cultured overnight inthe presence of each of the 11 hit compounds, in dose responses ranging30 μM to 300 nM. The samples were plotted as Annexin V vs 7-AAD andquadrant-gated. Annexin V⁺, 7-AAD⁻ events are indicative of earlyapoptosis, in which phosphatidyl serine has flipped to the outer leafletof the cell membrane. Annexin V⁺, 7-AAD⁺ (double positive) eventsindicate cell and nuclear membrane permeabilization, marking lateapoptotic events or complete cell death. The double positive events werethe primary focus for this assay, and this is shown in FIG. 4.

Many of the compounds induced apoptosis in U937 cells in adose-dependent manner, with some compounds producing populations whichnearly entirely consisted of double positive events. Line graphs of thedose responses of the most efficacious compounds were plotted, and theirEC₅₀ values were determined to range from 3.29 μM to 283.14 μM (FIG. 4b). The compounds with the best EC₅₀ values, in order from lowest tohighest, were patulin, pyrithione zinc, parthenolide, cryptotanshinone,and dihydroartemisinin.

cAMP Efflux Inhibitors Caused Cell Cycle Arrest

To assess the effects of the hit compounds on U937 cell cycle, apropidium iodide (PI) staining assay was conducted. The 11 hit compoundswere tested in dose response, under the same conditions as the apoptosisexperiments. Samples were analyzed with flow cytometry for PI MFI (seeexample in FIG. 4A a), and the percentages of the populations withingates for each phase of the cell cycle are plotted in FIG. 4A b. Thiswas relevant, as increased intracellular cAMP is known to arrest cellsat G₁ ^(5,6). The most active compounds (artesunate, parthenolide,dihydroartemisinin, and cryptotanshinone) increased percentages of cellsin G₁ or apoptosis in a dose-dependent manner (FIG. 4A c).

cAMP Efflux Inhibitors Decreased Cell Proliferation

Because there was a possibility that the hit compounds may haveadditional debilitating effects on U937 cells, their influences onproliferation were investigated as well. To accomplish this,carboxyfluorescein diacetate succinimidyl ester (CFSE) was loaded intocells to bind to intracellular proteins and serve as a fluorescentmarker for cellular generation. Lower remaining CFSE values indicatehigh proliferative capacity. Post-staining, cells were grown in thepresence of compound, and samples were collected after 2, 3, and 4 dayincubations. Samples were measured for remaining CFSE fluorescence byflow cytometry. Analysis revealed decreased cell fluorescence,indicating cell division. Some compounds (patulin, parthenolide,dihydroartemisinin) showed arrest of cell division within 48 hr, atconcentrations of 3 μM, while control condition cells continued toproliferate (FIG. 5A).

cAMP Efflux Inhibitors Decreased Cell Viability and Showed Cancer CellSpecificity

To validate increased intracellular cAMP as a target for inducingleukemic cell death, the hit compound effects on cell viability weremeasured with Cell TiterGlo®. The hit compounds were tested with U937cells, and several showed more potency than the positive control MK-571(FIG. 6A a, i). This assay was repeated with the top 7 compounds (ART,DHA, PTH, PLN, PZ, CQ, CTS) in 6 B-lineage ALL cell lines (see Table 1).These cell lines showed more sensitivity than the U937 cells to severalof the selected compounds (FIG. 6Aa, iii-viii), but showed decreasedsensitivity to the artemisinin derivatives, artesunate anddihydroartemisinin. The EC₅₀ values for the hit compounds with each cellline ranged from low nanomolar to ˜200 μM (FIG. 6A b). It can be noted,however, that the compounds which consistently decreased viability thebest were “ranked” the same across the 6 B-ALL lines: pyrithione zinc,patulin, parthenolide, clioquinol, cryptotanshinone.

These results led to the examination of potential selectivity of the hitcompounds for decreasing viability in leukemic cells over healthy cells.Therefore, the same viability assay was repeated with healthy humanperipheral blood mononuclear cells (PBMCs). Comparison of the compoundswith all 8 cell types tested show PBMC responses at higherconcentrations than the tested cell lines, indicating compoundselectivity for the cancer cells (FIG. 6A a, ii). Calculated EC₅₀ valuesindicate that for most of the tested compounds, leukemic cells requiredconcentrations an order of magnitude or lower than was necessary for thePBMCs to be efficacious (FIG. 6A b). This is expected, because normalPBMCs would not have an established system for cAMP efflux, andtherefore would not be as sensitive to our identified efflux-inhibitingcompounds. The exception was pyrithione zinc, which is a known biocidalcompound that in fungi inhibits membrane transport and decreasescellular ATP²⁰, and in human skin cells, decreases ATP viapoly(ADP-ribose) polymerase involvement as well as DNA damage andupregulation of heat shock proteins²¹.

Leukemic Cell Lines have Different Abilities to Efflux cAMP

To study differences in leukemic cell cAMP efflux, human cell linesrepresenting different B-lineage ALL phenotypes (see Table 1 forinformation on genetic rearrangements for each line) were loaded withF-cAMP, and its efflux from the cells was studied. The cells wereincubated overnight: 1) at 4° C. to estimate the passive F-cAMP probe“leakage”, 2) at 37° C. to assess active removal of the probe, and 3) at37° C. in the presence of the positive control 100 MK-571, a previouslyreported inhibitor of cAMP efflux to estimate the “pump-dependent”component of the process. Because cells loaded with F-cAMP were washed,the resulting concentration gradient could have caused a “passive leak”of the probe. All 6 cell lines appeared to efflux F-cAMP, although todifferent extents (FIG. 7A a). After incubation at 4° C., the cells lost20-60% of their fluorescence from the initial F-cAMP loading. Incubationat 37° C. resulted in F-cAMP removal that ranged from ˜60% for Sup B15and MHH Call 3 cells, to ˜90% for 697 and Nalm-6 cells, with no apparentcorrelation between 4° C. and 37° C. samples. For example, Nalm 6 cellsshowed the smallest “passive leak” with the highest active proberemoval, and 697 cells exhibited a very significant “leakage”, with highactive probe removal comparable to Nalm 6 cells.

The cell lines also exhibited varied sensitivity to the effect ofMK-571. The responses ranged from 0-60%, wherein “0” corresponds to theabsence of F-cAMP efflux. For two cell lines, Sup B15 and MHH Call 3,the inhibitor completely blocked F-cAMP removal. For these cell lines,MK-571 blocked not only active probe removal at 37° C., but also effluxat 4° C., and therefore the fluorescent signal was equal to the initialcAMP probe loading value (FIG. 7A a). One possible explanation for thisphenomenon could be that the inhibitor is interfering mechanically withthe cAMP probe fluxes, for example, by blocking a “cAMP-specificchannel”. For 697 cells, MK-571 blocked F-cAMP removal close to thelevel of the “passive” 4° C. leakage, and for other cell lines theeffect was different.

Because the results appeared so diverse, the fluorescence values afterincubation with and without MK-571 were plotted against one another. Wefound a strong relationship between levels of the probe remaining in thecells at 37° C. alone and in the presence of the MK-571. Based on thesedata, the cell lines could be stratified into three groups whichindicated correlations between ability to actively efflux cAMP andability to block cAMP removal with MK-571 (FIG. 7A b). Group 1: Sup B15and MHH Call 3 removed only ˜60% of the probe without the inhibitor, andthe F-cAMP efflux was blocked completely by MK-571. Group 2: Reh andRS4; 11 cells removed 75-80% of the probe at 37° C., and ˜30-40% in thepresence of MK-571. Group 3: 697 and Nalm 6 cells removed more than 90%of the probe without MK-571, and 60-70% in the presence of MK-571 (FIG.7A b). Essentially, those cell lines which poorly effluxed cAMP werebest inhibited by MK-571, and those cell lines which actively removedmost of the incorporated F-cAMP were less inhibited by MK-571.

These variations indicate the possibility that in different cell lines,the cAMP removal system which is sensitive to MK-571 represents adifferent fraction of the overall cAMP-pumping machinery. For Sup B15and MHH Call 3, it represents a large portion of it, and therefore, theaddition of MK-571 was sufficient to fully block probe efflux. For theother cell lines, additional MK-571-insensitive mechanisms participatedin the removal of the probe, with the biggest fraction in Group 3. Thus,cell lines exhibited a large heterogeneity of the cellular machineryresponsible for the removal of the fluorescent cAMP analog.

We then tested our identified hit compounds with the 6 cell lines indose response under the same conditions as the HTS. FIG. 7A c shows theresults of the assay, and it is apparent that the abilities of thecompounds to inhibit cAMP efflux from the B-lineage cell lines varied.

This is underscored by the calculated EC₅₀ values determined from theassay (FIG. 7A d). However, these results were expected, consideringthat the B-lineage ALL cell lines had shown differing abilities toremove F-cAMP under normal conditions.

The Primary cAMP Transporter, ABCC4, is Differentially Expressed byDifferent Leukemic Cell Lines

cAMP is typically released by cells via ATP-binding cassette (ABC)transporters (also known as multidrug resistance proteins (MDRs orMRPs)). According to the FDA Transporter Database, two ABC transportershave been reported to transfer cAMP out of a cell: ABCC4 (MRP4) andABCC5 (MRP5)²². It should be noted that the affinity of ABCC4 for cAMPis ˜100× greater than that of ABCC5 (k_(m) values of 44.5 μM and 379 μM,respectively)^(22,23). Furthermore, while ABCC4 is expressed on theplasma membranes of CD34+ leukocytes, its expression decreasessignificantly upon cell differentiation¹². Therefore, we hypothesizedthat the presence of MRP4 on leukemic cells would correlate with theability to remove cAMP from the cells, as a mechanism for apoptoticescape.

ABCC4 phenotypes were determined for the B-lineage ALL cell lines (Table1). Primary anti-human ABCC4 antibodies and isotype controls were boundto fixed cells in matched samples. The calculated number ofMRP4-specific binding sites ranged from ˜100 ABCC4 sites on MHH Call 3cells to ˜10⁴ binding sites on RS4; 11 cells (FIG. 8). It is possiblethat other transporters and/or mechanisms may play additional roles inleukemic cell removal of cAMP.

The cAMP Efflux Inhibitors Increase Intracellular cAMP Accumulation andDecrease ATP

The results of the viability assay described above confirmed that theidentified hit compounds decreased intracellular ATP. To reaffirm thatthe decreases in leukemic cell viability were due to the hit compoundeffects on increasing cAMP, the Promega cAMP-Glo™ assay was performed tomeasure cAMP accumulation within the cells. The 6 aforementionedB-lineage ALL cell lines (Table 1), U937 cells, and PBMCs were eachincubated ˜24 hours with the hit compounds, in dose response, and thenluminescence was measured; in this assay, there is an inversecorrelation between cAMP levels and luminescence. These luminescencevalues were then transformed into cAMP concentrations for each sample(FIG. 9a ).

These results indicated a correlation between a decrease in cellviability and cAMP accumulation in some B-lineage ALL cell lines. Theefficacy of the compounds also “ranked” in the same order as they didwith the aforementioned assays, with pyrithione zinc, patulin,parthenolide, clioquinol, and cryptotanshinone resulting in the lowestEC₅₀ values. This relationship is further supported by the fact thatwhen the EC₅₀ values for both cell viability and cAMP accumulation wereplotted against one another, there were high coefficients ofdetermination (r² values) and slopes approximately equal to 1 (FIG. 9b). Therefore, the identification of compounds based on their abilitiesto inhibit cAMP efflux as a means to selectively treat leukemic cellshas been validated.

Discussion

Targeting cAMP Efflux

The HTS was an efficient, unbiased method to find compounds targeted toa cancer cell-specific functional, rather than physical, trait: cAMPefflux. The libraries screened contained off-patent, FDA-approved drugs,and can allow for faster turnaround time for potential translation toclinical therapeutic use of these compounds for patients withhematological malignancies. Our screen resulted in the identification ofseveral active compounds which caused inhibition of cAMP efflux andresulted in increased intracellular cAMP. These compounds when usedalone inhibited proliferation and viability, and induced apoptosis andcell cycle arrest in the U937 AML cell line. Moreover, these compoundsdecreased viability in B-lineage ALL cell lines, and the EC₅₀s of themost efficacious compounds ranked in the same order across cell lines:pyrithione zinc, patulin, parthenolide, clioquinol, cryptotanshinone.Importantly, when the hit compounds were tested on healthy human PBMCs,the measured EC₅₀ was much higher, indicating higher sensitivity ofleukemic vs. normal cells. These findings support our hypothesis, andgive validation to cAMP efflux as a cancer cell functional target.

Classically, cyclic nucleotide analogs or other cAMP-elevating agentssuch as all-trans retinoic acid have been used in treatment ofhematological malignancies to slow cell growth and differentiate cancercells. While modestly effective, these compounds had toxicity innon-cancerous tissues²⁴. Our approach also seeks to use a cyclicnucleotide to inhibit blood cancers, however, this is achieved byincreasing intracellular cAMP by preventing its efflux, which itself isa protective mechanism developed by malignant cells to evade typicalapoptotic signals. Because increased cAMP production and efflux is not atypical trait of healthy cells, the cAMP efflux inhibitors that we haveidentified have some selectivity for cancerous cells.

Of the potentially active compounds identified in our screen for cAMPefflux inhibitors, the majority of them were sesquiterpene lactones:parthenolide is derived from the feverfew (Tanacetum parthenium) plant;artemisinin and dihydroartemisinin are from Artemisia annua. Artesunateand artemether are semi-synthetic artemisinin derivatives. Severalidentified compounds were previously reported to exhibit anticanceractivity in a number of model systems (Table 2).

Parthenolide has been shown to prevent activation of the NF-κBpathway²⁷, and it is suspected that this occurs via inhibition of theIκB kinase²⁸⁻³⁰. Additionally, parthenolide inhibits the transcriptionfactor AP-1 and ERK by preventing phosphorylation^(29,31). In studieswith AML cells, parthenolide was shown to potentiate cell death whenused in combination with histone deacetylase inhibitors (HDACIs)³².Parthenolide's ability to inhibit the NF-κB pathway has also been shownto more strongly inhibit cancer stem-like cells than non-stem cellcounterparts^(33,34). Because cAMP also has these effects on the NF-κBpathway, this may reassert that parthenolide is indeed increasingintracellular cAMP. When in vitro B-lineage CLL cells were treated withparthenolide, in addition to inhibition of NF-κB, reactive oxygenspecies were generated, thus adding to its apoptotic effects³⁵. Theseeffects likewise occurred when parthenolide was administered to melanomacells. Interestingly, parthenolide treatment also resulted in areduction of ABCB5-positive cells and those malignant cells whichremained had decreased proliferative ability³⁶.

Dihydroartemisinin is an antimalarial drug which has previously beenshown to selectively induce apoptosis in a myriad of cancer celllines³⁷⁻⁴² (see also Table 2). This cell death may occur byintrinsic^(38,42-45) and/or⁴⁶⁻⁴⁸ extrinsic^(37,40,49) apoptoticpathways. Additionally, dihydroartemisinin can decrease cellproliferation^(43,46,48,50,51), and cause cell cycle arrest at either G₁⁴³ or G₂/M^(42,45,46,52). One major mechanism by whichdihydroartemisinin may achieve these effects is through its binding toiron⁴⁷, which results in generation of reactive oxygen species(ROS)^(38,39,41,44,48-50) and downregulation of the transferrinreceptor^(50,52,53). This drug also inhibits NFκB^(43,46,54) andMAPK/ERK signaling^(40,47,55). Furthermore, dihydroartemisinin inhibitsVEGF and decreases angiogenesis^(51,52,56-59).

In lung adenocarcinoma, DHA causes ER stress and translocation of Bim tothe ER, but induced apoptosis is not dependent on these factors⁶⁰.

DHA inhibits MEK/ERK pathway and causes downregulation of Mcl-1, butonly apoptosis is caspase-dependent⁵⁵.

DHA shows selectivity for prostate cancer vs normal cells, inhibitsPI3K/Akt and MAPK/ERK pathways, causes apoptosis via extrinsic(upregulated expression of death receptor 5) mechanisms⁴⁰.

DHA is selectively cytotoxic to glioma vs normal cells, by inhibitingHIF-1α (thereby decreasing survival factors like VEGF) and increasingROS production due to interactions with iron⁴¹.

DHA is selective for ovarian cancer cells in induction of G₂ cell cyclearrest and apoptosis by decreasing expression of anti-apoptotic proteins(Bcl-2 and Bcl-x_(L)) and increasing expression of pro-apoptoticproteins (Bax and Bad)⁴².

-   -   In hepatocellular carcinoma DHA induces G2/M arrest via        inhibition of cyclin B and CDC25C, depolarization of        mitochondrial membranes, decrease in Mcl-1, increase in Noxa and        Bak, apoptosis involved p53 and caspases 3 and 9 (intrinsic        pathway)⁴⁵.

Artesunate is a phytoconstituent obtained from the Chinese medicinalherb Artemisia annua. It has a sesquiterpene lactone structure with aninternal peroxide bridge, which provides a different structuralprototype compared to classical antimalaria drugs such as e.g.chloroquine, quinine, proguanil or sulfadoxine. Semisyntheticderivatives of artemisinin; artesunate, artemether, and arteether; havebeen developed. After oral administration of artemisinin,deoxyartemisinin, deoxydihydroartemisinin, 9,10-dihydrodeoxyartemisininand a metabolite named ‘crystal 7’ were identified in human urine. Allfour metabolites are inactive due to the lack of the endoperoxidebridge. Artesunate is hydrolyzed within minutes to its activemetabolite, dihydroartemisinin, which is considered to be responsiblefor the antimalarial activity.

Patulin is a mycotoxin which is produced by Aspergillus or Penicillium,and is most often found in moldy fruit, particularly apples, and thismolecule binds to and forms adducts with sulfhydryl groups⁶¹.

-   -   In vivo (healthy male mice), induces apoptosis via DNA damage,        lipid peroxidation, and a decrease in amount of glutathione        (GSH)⁶². Showed selectivity for brain, liver, kidney over        bladder.    -   Increases DNA damage, ERK1/2 phosphorylation (associated with        extracellular signaling) and increases expression of early        growth response gene-1 in MDCK (canine kidney), HEK293, and        PBMCs⁶³. Sustained ERK activation can cause cell        differentiation.    -   It has been described that cAMP efflux via ABCC4 is dependent on        intracellular glutathione (GSH), and that decreased GSH leads to        decreased cellular export of cAMP⁶⁴. Therefore, it makes sense        that cell exposure to patulin, has been shown to decrease GSH in        other studies⁶² would be identified as a cAMP efflux inhibitor        in our HTS.    -   CHO-K1 cell cytotoxicity was induced by ROS, which led to lipid        peroxidation; also increased production of malondialdehyde⁶⁵.    -   Induces unfolded protein response signaling, including the        PERK/eIF2α/CHOP apoptotic and IRE1/XBP1 adaptive arms of the        response, and also caused activation of BH3-only genes and        apoptosis⁶⁶.    -   In Caco-2 colon cancer cells, patulin disrupted tight junctions        by downregulating ZO-1 (via increased        phosphorylation→degradation) and claudin-4⁶⁷.    -   In human keratinocytes, patulin decreased autophagosome        degradation, leading to increased p62 in a pro-survival role:        ROS generation, UPR, ERK1/2 phosphorylation, and inhibition of        BAD⁶⁸.    -   In colorectal cells, patulin caused cell cycle and growth arrest        at G₂/M, and ROS generation caused by the compound was        responsible for ATF3 expression and apoptosis⁶⁹.    -   In HEK293 and HL-60 cells, patulin caused lipid peroxidation and        ROS generation. This ROS generation influenced ERK1/2 signaling,        and vice versa⁷⁰.

Cryptotanshinone is a plant-derived (Salvia miotiorrhiza) compound whichexhibited cAMP efflux inhibition and coincided with decreased bloodcancer cell viability. While there is evidence that this compound may beselective for treatment-resistant malignant cells^(71,72), it should benoted that in our experiments, the EC₅₀ for decreased PBMC viabilitywith cryptotanshinone was only ˜12 which is a lower concentrations thatwere determined for some of the tested B-lineage ALL cell lines (SupB15, Reh, Nalm 6, MHH Call 3). However, cryptotanshinone has been shownto induce cell cycle arrest, decrease proliferation, and induceapoptosis in a myriad of cancer types; the efficacy of these effectsseem to vary based on metastatic capacity and type of cancer^(73,74). Itis hypothesized that cryptotanshinone can prevent the expression ofvital cell components such as cyclin D1^(71,75,76) and Bcl-2^(71,77). Itcan also stimulate the extrinsic pathway of apoptosis by upregulatingexpression of TRAIL receptor 2⁷², and activate caspase 3 and Bax⁷⁷. Someof the antiproliferative effects of cryptotanshinone may be due toinhibition of mTOR signaling, resulting in decreased expression ofcyclin D1 and eukaryotic initiation factor 4E, and decreasedphosphorylation of retinoblastoma (Rb) protein phosphorylation⁷⁵. Inmelanoma cells, cryptotanshinone caused increased expression of p53,Chk1, and Chk2⁷³. Cryptotanshinone is also known to have antitumoreffects by inhibition of the STAT3 pathway^(76,78). It is interesting tonote that studies to determine the bioavailability of cryptotanshinonein the gut found that the drug may be a substrate of the P-glycoprotein(ABCB1) transporter⁷⁹.

Clioquinol is an antimicrobial drug that has recently been tested as apotential anticancer therapeutic. It is binds to divalent metal ions,and can serve as a chelator or ionophore. Consequently, there aremultiple potential mechanisms of action in terms of clioquinol'santi-blood cancer effects. As a zinc chelator, clioquinol has been notedto fit into the binding pockets of histone deacetylases, therebyinhibiting their activities⁸⁰. This has resulted in upregulation oftumor suppressor genes, such as p21, p27, p53, and additionally, incessation of proliferation and apoptosis⁸⁰. As a transition metalionophore, clioquinol increases intracellular zinc concentrations andleads to apoptosis⁸¹, and it is suspected that this may be due to downregulation of NF-κB signaling⁸¹⁻⁸³, and increasing lysosomal [zinc]⁸².Further support for its ability to inhibit NF-κB may be inferred fromthe fact that cyclin D1, a downstream gene targeted by NF-κB, and majorplayer in the regulation of the cell cycle, is decreased after exposureto the clioquinol⁸⁴. Clioquinol is also able to bind with copper, andtherefore can inhibit proteasome activity and induce apoptosis⁸⁵⁻⁸⁷;these effects may produce signals to activate macrophages to releaseTNF-α, further increasing cancer-cell specific activity⁸⁸. Currently,clioquinol is being tested in a Phase I clinical trial for potentialtranslation into a treatment for hematological malignancies⁸⁹.

8-quinolinol, a compound closely related to clioquinol has been shown tobe especially effective in decreasing the proliferation of breast cancerstem cells over regular breast cancer tumor cells, and it is thoughtthat its mechanism of action is through inhibition of NF-κBactivation⁹⁰.

cAMP Regulation

cAMP plays several significant regulatory roles within cells. Therefore,it is very plausible that cancer cells, especially those resistant totreatment, have developed adaptations to supersede conditions by whichintracellular cAMP would result in differentiation or apoptosis. cAMPhas been shown to impede tumor suppressor p53 from accumulating, thuscells have less response to DNA⁹¹. Also, cAMP can inhibit and promoteNF-κB activity⁹². Constitutive activation of NF-κB is an important traitin leukemic stem cells^(90,93). NF-κB is pro-survival (hence oftenassociated with cancer), inhibition is pro-apoptotic. cAMP inhibits p53,so prevents apoptosis⁹⁴. It has been documented that increasedintracellular cAMP inhibits the ability of NF-κB to facilitatetranscription⁹⁵.

In some studies with cancer cell lines, increased cAMP has been shown toinhibit proliferation and increase c-KIT expression⁹⁶. These effectsseem paradoxical, as c-KIT is known to positively regulate cell growth.Elevated c-KIT had no effect on cell growth⁹⁶. cAMP stimulation orinhibition of MAP kinase/ERK is cell type-specific⁹⁷. In most tumor andhematological cells, cAMP inhibits ERK, and results in cell cycle arrestand ablation of proliferation^(98,99).

GPCRs, Adenylyl Cyclase (AC), PDEs as Targets for cAMP Level Modulation

Targeting cancer and specifically hematological malignancies through a“pathway-dependent approach” that consists of different means ofelevating cAMP is considered a valuable option for novel therapeuticdevelopment¹⁰⁰. Starting with the increasing of AC activity using GPCRligands, antagonists for G_(i)-coupled receptors or agonists forG_(s)-coupled receptors, direct AC activators, PDE inhibitors and drugsmodifying cAMP downstream signaling, all represent various approaches tothe stimulation of cAMP accumulation. It appears that every potentialstep of cAMP synthesis, degradation, and downstream signaling was takeninto a consideration¹⁰⁰⁻¹⁰². However, if efflux of cAMP from the cell isthe major mechanism of decreasing the cAMP concentration, therapiesbased on other pathway steps will be obsolete^(11,100) Our data suggestthat targeting cAMP efflux could be an efficient way to raise cAMP incertain types of cancer. The relative efficiency of this approach woulddirectly depend on the expression and the activity of the target pumpsthat is anticipated to be cell- or patient-specific.

cAMP Efflux Mechanism

The two cAMP efflux proteins, ABCC4 and ABCC5²², are unique among theABC transporters, and lack much structural similarity to other multidrugresistance proteins and one another⁶⁴. ABCC4 is non-vital for survival,as evidenced by normal phenotypes of MRP4−/− mice. However, this proteinis especially important in nucleotide sensitivity of the bone marrow,thymus, spleen, and intestine¹⁰³. In comparison to normal hematopoieticcells, blood cancer cells express more ABCC4¹⁰⁴. However, xenografts ofthese cancer cell lines in the spleen and thymus are particularlysensitive to inhibition of ABCC4 substrate (mitoxantrone) efflux byMK-571¹⁰⁴. The fact that ABC transporters are upregulated in stem-likecells may suggest that these cells require the removal of cAMP or otherstructurally-related compounds from the cytoplasm in order to remain ina pluripotent state. The other possibility is that the metabolicspecificity of these cells requires active pumping of certainmetabolites¹⁰⁵.

The results of our ABCC4-labeling assay did not determine any clearcorrelations between the cAMP efflux ability and ABCC4 expression in thecell lines. It is possible that other ABC transporters capable of cAMPefflux are present. ABCC5 or ABCC11 are good candidates for thisrole^(23,106). A difference in the pump activity is the other possibleexplanation. The increased concentrations of cAMP in urine of leukemiapatients may be explained by efflux from ABCC4¹⁰⁷.

However, known substrates/inhibitors of ABCC4 (sulindac, diclofenac,topotecan) were also in the libraries used for the preliminary screen(sometimes in duplicate), yet did not come out as hits. This may meanthat inhibition of the pumps may potentially be substrate- orcondition-dependent. Future studies will need to focus more on thesetransporters to implicate their roles in cAMP efflux.

Additionally, cancer cells with stem cell-like characteristicspotentially express higher levels of other ABC transporters, e.g. ABCG2,which represent an inherent “molecular determinant” of the“side-population” analysis that is commonly used to identify stemcells^(13,108). These cancer stem cells are associated with higherresistance to typical cancer therapeutics¹⁰⁹. We anticipate thatidentification of drugs that could inhibit these transporters wouldallow for more selective targeting of resistant cancers and betterpatient outcomes. For example sildenafil, a PDE 5-specific inhibitor,decreases ABCG2- and ABCB1-mediated drug efflux¹¹⁰. This meritsinvestigation into the effect of identified compounds on other ABCtransporters.

TABLE 2 Most active identified cAMP-efflux inhibiting compounds CompoundCancers(s) treated Patulin kidney⁶³, oral squamous cell carcinoma⁶⁶,glioblastoma¹¹¹, colon⁶⁷, colorectal⁶⁹, hematological⁷⁰ Parthenolidemelanoma^(27,36), cervical^(29,30), hematological^(32,34,35), breast³³,(cystic fibrosis^(28,31)) Artesunate ovarian³⁷, cervical³⁹ Dihydro-ovarian^(37,42), breast⁵⁰, liver^(45,50), melanoma³⁸, artemisininpancreatic^(43,49,54,57,112), lung^(48,59,60), cervical³⁹,hematological^(44,47,51,52,55,58), prostate⁴⁰, glioma⁴¹, osteosarcoma⁴⁶Clioquinol hematological^(80,81,86,89), breast^(84,85), HeLa⁸⁸,prostate^(82,83,87) Cryptotanshinone hematological^(71,76),prostate^(74,75,78), breast^(75,77), melanoma^(72,73), lung⁷²,rhabodomyosarcoma⁷⁵

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What is claimed is:
 1. A method of reprogramming a population of cancercells to reestablish an apoptotic escape, comprising exposing saidcancer cells to an effective amount of at least one compound selectedfrom the group consisting of

harmalol, artemisinin and artemether or a pharmaceutically acceptablesalt or stereoisomer thereof.
 2. The method according to claim 1 whereinsaid population of cancer cells resides in a patient.